Isolex 300i Research Articles

Comparison of two T-cell depletion methods of grafts from alternative donors for allogeneic hematopoietic cell transplantation of patients with fanconi anemia.

Background & Aim Our program has used 2 T-cell depleted HSCT approaches for patients with FA for transplants from donors other than HLA matched siblings: 1) the Isolex 300i Magnetic Cell Selection System (Baxter) + sheep RBC depletion (E-) (TCD1) was used to achieve an approximate 5 log10 depletion of CD3+ cells, and (2) the CliniMACS device (Miltenyi; TCD2) was used as a sole TCD method with an approximate 4.5 log10 depletion of CD3+cells. Methods, Results & Conclusion We retrospectively reviewed charts of consecutive patients with FA who received a first HCT from alternative donors. We identified 42 patients with aplastic anemia, MDS or AML, 24 who received TCD1 (1999-2009) and 18 who received TCD2 (2010-present). Most patients in TCD1 received a TBI-based conditioning, while most in TCD2 received busulfan-based conditioning. Donors were matched unrelated, mismatched unrelated, or mismatched related. Because of the differences in the treatment periods, conditioning regimens, and disease breakdown, we analyzed the data specifically related to the T-cell depletion. Distributions of continuous variables were compared using a Mann-Whitney-Wilcoxon test, while binary and categorical variables were compared using a Fisher's exact test. Grafts were G-CSF mobilized peripheral blood progenitor cells. T-cell depletions were performed as described. Table 1 provides demographics, cell doses and hematologic recovery data for TCD1 and TCD2. Median CD34 cell doses of the grafts did not differ significantly while median CD3 dose did (N=0.048). All but one patient engrafted. Two patients receiving grafts from mismatched unrelated donors developed grade 2-4 acute GvHD in TCD1, none in TCD2. Neutrophil engraftment did not significantly differ while platelet engraftment was more rapid with TCD1 (N=0.0045). CD4 recovery (Abs CD4 >200) and PHA recovery (50th%ile) differences were comparable (NS). In conclusion, no difference were noted between the Isolex E- T-cell depletion and the CliniMACS CD34 selection system, except for a more rapid platelet reconstitution for the CliniMACS column. There was no difference in risks of graft rejection and GvHD, and immune reconstitution was comparable in both cohorts. Future plans include the comparison of these methods to the TCR alpha beta depletion in this patient population.

Rash in Patients after T-Cell-Depleted Peripheral Blood Stem Cell Transplantation: Eosinophilia and Pruritus Do Not Distinguish Acute Graft-Versus-Host Disease from Drug Rash

Background: The incidence and severity of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) is decreased with T-cell depletion. However, ~14% of these T-cell depleted (TCD) recipients will have aGVHD with skin involvement (Barba et al. 2017). Furthermore, alloHSCT recipients are at increased risk for drug eruptions and infectious exanthems (Byun et al. 2011). While histopathologic differences have been reported between aGVHD and non-aGVHD rash in TCD recipients (Fischer et al. 2015), skin biopsies alone are insufficient to determine rash etiology. As such, distinguishing inflammatory non-aGVHD rashes from aGVHD of the skin after TCD HSCT remains challenging and relies on clinical presentation. While peripheral eosinophilia is seen in both aGVHD and drug hypersensitivity, rashes that present with concomitant eosinophilia after HSCT are often suspected to have a drug-induced etiology. We sought to assess the incidence and features of aGVHD and non-aGVHD rashes within 1 year after TCD alloHSCT, as well as common etiologies of non-aGVHD rash. These findings may guide clinicians in earlier diagnosis and management of non-aGVHD rash.Methods: Using a clinical research database, 243 adult patients were identified who received alloTCD peripheral blood stem cell transplantation (PBSCT) at a single institution between 2008 and 2011. All patients had CD34+ hematopoietic progenitor cells selected using: the Isolex 300i Magnetic Cell, followed by additional T-cell rosetting with neuraminidase-treated sheep erythrocytes or using the CliniMACS CD34+ Reagent System (Table 1). Given decreased incidence of aGVHD with Isolex versus CliniMACS (Barba et al. 2017), we established Isolex and CliniMACS TCD groups and assessed aGVHD and non-aGVHD rash within these two CD34+ selection modalities. To identify non-aGVHD patients with skin rash, charts were reviewed from date of HSCT through 1 year post HSCT by review of dermatology visit notes or by extraction of International Classification of Diseases-9 (ICD-9) codes for skin lesion or rash (Table 2); skin infections and chronic GVHD rashes were excluded. Acute GVHD was diagnosed with histological confirmation when clinically indicated. Rash characteristics, including pruritus and peripheral eosinophilia at onset of rash, were collected from charts of both non-aGVHD and aGVHD rash patients.Results: Among 243 TCD PBSCT transplant recipients, 152 patients (63%) were identified with skin rash within 1 year after HSCT. Of these patients, 43 had aGVHD rash and 109 had non-aGVHD rash. The majority of aGVHD rashes had skin stage III aGVHD regardless of CD34+ selection method (Table 3). For patients with non-aGVHD rash, etiologies included inflammatory conditions (Table 4). TCD by Isolex led to non-aGVHD rash development at a median onset of 60 days and aGVHD rash at a median onset of 71 days; while TCD by CliniMACS led to non-aGVHD rash development at a median onset of 56.5 days and aGVHD rash at a median onset of 75 days. Of patients who had recorded complete blood counts at onset of rash, elevated percent eosinophilia (% eosinophils >7%) was present in 10% of Isolex and 5% of CliniMACS non-aGVHD rashes versus 3% of Isolex and 4% of CliniMACS aGVHD rashes. Peripheral eosinophilia was not associated with aGVHD versus non-aGVHD skin rash post TCD HSCT (p≥0.99) nor when separated by CD34+ selection method (Isolex p=0.673; CliniMACS p≥0.99). While non-aGVHD skin rash patients had higher incidence of pruritus compared to aGVHD skin rash, pruritus was not a significant predictor of aGVHD versus non-aGVHD skin rash (p=0.20) nor when separated by CD34+ selection modality (Isolex p=0.188; CliniMACS p=0.469).Conclusions: In our case series of 243 TCD PBSCT recipients of whom 152 had skin rashes, over three-quarters of all non-aGVHD skin rashes with clear etiologies were attributed to drug eruptions. Our results suggest that the commonly utilized feature of peripheral eosinophilia may not be helpful in differentiating between aGVHD and drug rashes after TCD alloHSCT. Additionally, pruritus at rash onset was not helpful in distinguishing cause of rash as due to aGVHD or non-aGVHD after TCD alloHSCT. Based on these data, if clinical scenario supports GVHD associated rash, the presence of peripheral eosinophilia or pruritus should not delay initiation of therapy for GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Importance of Positive Immunomagnetic Cell Separation Prior to Autologous Hematopoetic Stem Cell Transplantation for Advanced Stage Lymphomas

Abstract We present the method of immunomagnetic stem cell separation with the ISOLEX 300i device (Isolex® 300i Magnetic Cell Selection System, Nextell Therapeutics Inc. Irvine California 21618 USA) and the results obtained using this method in patients admitted to the Hematology and Bone Marrow Transplantation Clinic of Tîrgu Mureş, Romania. Cell selection has a great importance in separating stem cells from tumor cells, therefore contributing to the success of autologous stem cell transplantation.

Final product composition after ex-vivo T-cell reduction: Miltenyi CliniMACS versus Baxter Isolex 300I in a large cohort of allogeneic trans-plant patients

Final product composition after ex-vivo T-cell reduction: Miltenyi CliniMACS versus Baxter Isolex 300I in a large cohort of allogeneic trans-plant patients

Ex-Vivo T Cell-Depleted Versus Unmodified Allografts in Patients with Acute Myeloid Leukemia in First Complete Remission: A Comparison of Outcomes in Two Institutions

Abstract 2013Allogeneic stem cell transplantation (ASCT) with T-cell depleted (TCD) allograft has been used to decrease the risk of graft-versus-host disease (GVHD). How outcomes of ASCT with TCD grafts compares to transplant with unmodified grafts in the context of ablative, reduced toxicity conditioning with busulfan /fludarabine is unknown. We sought to compare ASCT with and without TCD grafts, by studying the outcomes of pts with acute myeloid leukemia (AML) in first complete remission (CR1) who had TCD allografts at Memorial Sloan Kettering Cancer Center (MSKCC) with those who had unmodified allografts at MD Anderson Cancer Center (MDACC).Transplants at MSKCC (N=115) and MDACC (N=181) were performed between 2001 and 2010. Preparative regimens at MSKCC included: 1) Hyperfractionated total body irradiation (HFTBI) 1375 cGy over 4 days, thiotepa 5 mg/kg/day × 2 days & fludarabine 25 mg/m2/day × 5 days (n=29); 2) HFTBI 1375 cGy over 4 days, thiotepa 5 mg/kg/day × 2 days & cyclophosphamide 60 mg/kg/day × 2 days (n=25); or 3) busulfan 0.8 mg/kg intravenously every 6 hours for 10 doses(n=42) or 12 doses (n=19), melphalan 70 mg/m2/day × 2 days & fludarabine 25 mg/m2/day × 5 days (total n=61). Peripheral blood (PB) grafts underwent CD34+ cell selection using the ISOLEX 300i system, followed by sheep red blood cell (sRBC)-rosette depletion (n=85); or CD34+ cell selection using the CliniMACS system alone (n=22). Bone marrow (BM) grafts were T cell depleted by sequential soybean lectin agglutination and sRBC-rosette depletion (n=8). No additional GVHD prophylaxis was administered at MSKCC.Fludarabine 40 mg/m2 and busulfan 130 mg/m2 intravenously once daily for 4 days were used for conditioning at MDACC, with tacrolimus and mini-methotrexate for GVHD prophylaxis. Pts with unrelated or HLA-mismatched donors treated at MDACC and MSKCC received equine (total 60 mg/kg) or rabbit (total 2.5–5 mg/kg) anti-thymocyte globulin. Cytogenetic risk stratification considered complex cytogenetics, -5, -5q, -7, -7q, 11q23 aberrations, inv(3), t(3;3), t(6;9), and t(9;22) as poor risk.Pt characteristics and outcomes are summarized in the Table. TCD recipients were more likely to be older, receive a mismatch transplant, and have PBSC used as a graft source. Overall, only one pt, who had received an unmodified graft, experienced primary graft failure. Eleven (10%) and 33 (18%) pts experienced grade ≥2 acute GVHD within 100 days after transplants with TCD and unmodified grafts, respectively (p=0.05). Three-year relapse-free and overall survival rates were 58% and 57% in pts who received TCD grafts, and 60% and 66% in pts who received unmodified grafts (P=NS; Figure). There was a trend towards lower early non-relapse mortality among pts who received unmodified grafts (3% vs. 8% at 100 days, p=0.07). Of age, sex, secondary disease, risk status, donor, and graft type, secondary AML was the only risk factor found to significantly influence overall survival (HR: 1.7, 95% CI: 1.1–2.8). Survival outcomes were still comparable between recipients of TCD and unmodified grafts after adjusting for secondary AML. None of these factors significantly predicted relapse-free survival. Conclusion:Myeloablative ASCT with TCD or unmodified allografts lead to comparable outcomes in this high-risk cohort of AML pts in CR1 with a lower acute GVHD rate observed after TCD transplants.CharacteristicsFrequency, n (%)TCD, N=115Unmodified, N=181pTime CR1 to transplant, days*83 (12–304)97 (8–455)0.040Age, years*52 (19–71)48 (18–63)<0.001>50 years66 (57)76 (42)0.010Sex = Female58 (50)90 (50)NSEtiologyde novo60 (52)144 (80)<0.001Secondary38 (33)24 (13)Therapy-related17 (15)13 (7)Cytogenetic risk statusNSGood1 (1)2 (1)Intermediate72 (63)103 (57)Poor42 (37)76 (42)Donor typeMRD56 (49)103 (57)MUD32 (28)64 (35)MM27 (23)14 (8)<0.001Graft typeBM8 (7)57 (32)<0.001PB107 (93)124 (68)Outcome (95% CI)3-year RI18% (12%–27%)25% (19%–33%)NS100 day NRM8% (4%–15%)3% (1%–7%)0.071-year NRM18% (12%–27%)13% (9%–19%)NS3-year NRM24% (17%–34%)16% (11%–23%)NS3-year RFS58% (47%–67%)60% (51%–67%)NS3-year OS57% (47%–67%)66% (58%–74%)NSTCD: T-cell depleted, CR1: First complete remission, MRD: Matched related donor, MUD: Matched unrelated donor, MM: HLA-mismatch donor, BM: Bone marrow, PB: Peripheral blood, RI: Relapse incidence, NRM: Non-relapse mortality, RFS: Relapse-free survival, OS: Overall survival.*: Median (range). [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Immunologic Myeloablation After Haploidentical Peripheral Stem Cell Transplantation for Severe Combined Immunodeficiency

RATIONALE: Myeloid reconstitution after haplo-identical HLA mismatched related (HIMR) transplants for treatment of severe combined immunodeficiency (SCID) is rarely described. The mechanisms for donor myeloid engraftment in unconditioned HIMR are unknown. METHODS: We describe the outcomes of two patients with T-B + NK- SCID (γc mutation) who underwent stem cell transplantation using peripheral stem cell (PSC) mobilization (CD34 positive selection using Isolex 300i system) from HIMR maternal donors without pre-transplant conditioning. RESULTS: Both patients developed grade II Graft Versus Host Disease (GVHD) 30-60 days after transplant which required treatment with glucocorticosteroids. In patient 1 transfer of maternal antineutrophil antibodies were identified on post-transplant day 11. Two months post-transplant he demonstrated >95% donor chimerism for both lymphoid and myeloid lineages. Patient 2 developed persistent neutropenia, not responsive to granulocyte colony-stimulating factor, coinciding with GVHD. Bone marrow biopsy revealed absent myeloid precursors. After displaying donor T cell chimerism three months after his initial transplant a second PSC mobilized collection from his mother was performed. Thirty days after his second transplant he demonstrated >90% maternal chimerism with resolution of neutropenia. CONCLUSIONS: Two different mechanisms of myeloid ablation are suggested: antibody mediated and GVHD. In the first case successful engraftment of lymphoid and myeloid cells of donor origin has occurred. We speculate that myeloid ablation allowed for successful donor myeloid engraftment. In the second case initial studies after the second transplant indicate correction of neutropenia in the presence of donor T cell chimerism, suggesting the chimeric state allowed for maternal myeloid engraftment.

Therapeutic Potential of Intramyocardial Transplanted CD34+ Cells in Myocardial Ischemia

Baxter's Isolex 300i Magnetic Cell Selection System isolates patient's CD34+ cells from peripheral blood. The mononuclear cell fraction is incubated with anti‐CD34 antibodies and captured using sheep anti‐mouse (SAM) IgG coated beads. The therapeutic potential of intramyocardial transplanted, autologous, SAM‐isolated CD34+ cells is under clinical investigation. Due to the theoretical potential for prion transmission and limited supply of the source sheep, a rat anti‐mouse (RAM) IgG coated bead has been developed. In vitro, RAM and SAM were comparable in their ability to isolate CD34+ cells. CD34+ cells demonstrated comparable yield, purity, viability, apoptosis, and cell subsets. CD34+ cells exhibited comparable clonogenic potential and migratory capacity in a dose‐dependant manner to VEGF and SDF‐1. In vivo, intramyocardial transplantation of RAM‐isolated human CD34+ cells improved cardiac performance in an athymic rat, left anterior descending coronary artery occlusion model. Echocardiographic evaluation demonstrated improvements in left ventricular fractional shortening (p&lt;0.001) and left anterior wall thickening (p&lt;0.01), and decreased ventricular fibrosis, similar to that reported by Kawamoto et al (2003) in support of the IND for SAM. In conclusion, these data suggest equivalent preclinical efficacy of RAM and SAM‐isolated human CD34+ cells. Baxter provided all research support.

Long-Term Follow-Up of Patients with Non-Hodgkin Lymphoma Following Myeloablative Therapy and Autologous Transplantation of CD34+-Selected Peripheral Blood Progenitor Cells

Graft engineering by CD34(+) selection of peripheral blood progenitor cells (PBPC) has been used in non-Hodgkin lymphoma (NHL) with the aim to reduce relapse related to tumor cells within the graft. From September 1995 to January 2000, 39 patients with newly diagnosed (n = 31) or relapsed (n = 8) NHL were treated in our institution with myeloablative therapy followed by CD34(+) selected autologous PBPC transplantation. Thirty-one patients were diagnosed with follicular lymphoma, and eight patients with mantle-cell lymphoma. All patients had advanced disease (26% of patients stage III and 74% stage IV, Ann Arbor classification). Induction therapy resulted in a complete remission in 17 patients and a partial remission in 22 patients. PBPC were mobilized after cytotoxic chemotherapy with granulocyte colony-stimulating factor support. CD34(+) selection was performed using immunomagnetic beads (Baxter Isolex 300SA or 300i Magnetic Cell Separation System). Most patients (85%) received total body irradiation and high-dose cyclophosphamide as myeloablative regimen. Twelve patients also received rituximab 375 mg/m(2) before radiation and before the start of the cyclophosphamide treatment. The mean CD34(+) cell number for transplantation was 6.5 x 10(6) CD34(+) cells/kg of body weight. Platelet recovery (>20,000/microl median on day 13) and leukocyte recovery (>1,000/microl median on day 12) were within expected range. The estimated median follow-up was 47 months. The probabilities of freedom from progression, overall survival, and event-free survival 4 years after transplantation were 96%, 90%, and 87%, respectively, for patients with follicular lymphoma and 42%, 63%, and 33%, respectively, for patients with mantle-cell lymphoma. Risk factors for relapse were age and extranodal manifestation of disease. The rate of lethal infections in the 12-month follow-up period was 8%. We conclude that CD34(+) selection of autologous transplants following myeloablative therapy is feasible and results in long-term remission in the majority of patients, but the procedure is probably related to a higher rate of lethal infections.

A clinical‐scale expansion of mobilized CD34+ hematopoietic stem and progenitor cells by use of a new serum‐free medium

The autologous transplantation of CD 34+ cells expanded ex vivo in serum-free conditions dramatically reduces post-myeloablative neutropenia in myeloma patients. In our cell therapy unit, cells for this clinical assay have been expanded under GMP with serum-free Irvine Scientific (IS) medium with stem cell factor (SCF), granulocyte-colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor (MGDF; 100 ng/mL, respectively). Because this clinical-grade IS medium is no longer available, a new serum-free medium, Maco Biotech HP 01 (Macopharma), was evaluated. Purified CD 34+ cells (Isolex 300i, Baxter) from mobilized peripheral blood samples of myeloma patients were thawed, washed, and cultured, as for previous clinical assays. Twenty million CD 34+ cells were resuspended per 1 L of SCF-, G-CSF-, and MGDF-supplemented medium (HP 01 or IS), introduced into 3-L culture bags (AFC), and cultured for 10 days in 5 percent CO(2), at 37 degrees C, and at 100 percent humidity. A higher amplification of total nucleated cells (NCs) and colony-forming cells (CFCs) was obtained with HP 01 medium than with IS medium (42+/-16.6-fold vs. 20.5+/-5.9-fold for NCs and 26.7+/-7.4-fold vs. 15.5+/-2.5-fold for CFCs, respectively), whereas an increase in CD 34+ cells (3.5+/- 1.2-fold for HP 01 vs. 2.7+/- 1.5-fold for IS) was not significant. IS medium partially maintained SCID-repopulating cells (SRC), whereas the culture in HP 01 medium fully maintained the stem cell activity for 10 days. A higher frequency of CD 41+ cells after expansion in HP 01 than in IS medium was also observed. Maco Biotech HP 01 medium is suitable for clinical-scale expansion of CD 34+ cells with the SCF, G-CSF, and MGDF cytokine cocktail, permitting an intensive amplification of CFCs and maintenance of SRCs.

Hematopoietic Stem Cell Transplantation (HSCT) as Curative Therapy for Fanconi Anemia (FA) Patients with Acute Leukemia or Advanced Myelodysplasia (MDS).

Historically outcomes have been very poor for FA patients with advanced MDS or leukemia. It remains controversial as to whether HSCT is indicated for such patients and there is no consensus as to the optimal conditioning regimen in this setting. Traditionally, conditioning for FA patients has incorporated total body irradiation (TBI). Here, we report results of a pilot study in which we have substituted busulfan for TBI, designed for FA patients with one or more high risk features, identified following analysis of 42 previous consecutive URD FA transplants: advanced MDS or leukemia, age >18 years, or previous proven fungal or gram negative infection. Between 12/02–08/04 6 patients were enrolled, 5 with acute leukemia. Patient and disease characteristics are presented in Table 1. Conditioning consisted of busulfan (total 3.2mg/kg), cyclophosphamide (total 40mg/kg), fludarabine (total 140 mg/m2) and ATG (total dose 75mg/kg); GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. All patients received prophylactic voriconazole for one month prior to transplant. BM was T cell depleted with CD34 selection by Isolex 300i. Five out of six patients achieved neutrophil engraftment. Median time to an ANC>500 was 16 days (range: 11–20 days). One patient developed Grade I acute GVHD; no patient has developed chronic GVHD. The preparative regimen was well tolerated. Toxicities included Grade IV mucositis (n=1), VOD (n=2), hemorrhagic cystitis (n=1) and CMV pneumonia (n=1). Three patients are alive and in remission with a median follow-up of 575 days.Table 1AgeDiagnosisFANC groupRemission statusDonor sourceD+21 chimerismD+60 chimerismVital status5.9ALLBRCA2treated; CR5/6 related BM100% donor100% donoralive d+89421.7AMLAuntreated5/6 URD BM99.1% donor100% donordied resp. failure d+9920.8SAAAN/A5/6 URD BMinsufficient cellsN/Adied VOD d+246.6ALL,MDS,Wilm'sBRCA2ALL treated CR; MDS (7.5% blasts)6/6 URD UCB100% donor100% donoralive d+5757.1AMLAtreated; refractory4/6 UCB + 5/6 UCB100% donor #254.7% #2; 45.3% recipientdied AML relapse d+6017.3AMLAtreated; refractory5/6 UCB x266.7% #1; 31.3% #2; 2% recipient100% donor #1alive d+423Patient and disease characteristicsThese results suggest TBI is not required to achieve durable engraftment and leukemia control, busulfan 3.2 mg/kg is tolerable, and advanced MDS or acute leukemia does not preclude HSCT in FA patients.

Closed system generation of dendritic cells from a single blood volume for clinical application in immunotherapy

Dendritic cells (DC) used for clinical trials should be processed on a large scale conforming to current good manufacturing practice (cGMP) guidelines. The aim of this study was to develop a protocol for clinical grade generation of immature DC in a closed-system. Aphereses were performed with the Cobe Spectra continuous flow cell separator and material was derived from one volume of blood processed. Optimisation of a 3-phase collection autoPBSC technique significantly improved the quality of the initial mononuclear cell (MNC) product. Monocytes were then enriched from MNC by immunomagnetic depletion of CD19+ B cells and CD2+ T cells and partial depletion of NK cells using the Isolex 300I Magnetic cell selector. The quality of the initial mononuclear cell product was found to determine the outcome of monocyte enrichment. Enriched monocytes were cultured in Opticyte gas-permeable containers using CellGro serum-free medium supplemented with GM-CSF and IL-4 to generate immature DC. A seeding concentration of 1 x 10(6) was found optimal in terms of DC phenotype expression, monocyte percentage in culture, and cell viability. The differentiation pattern favours day 7 for harvest of immature DC. DC recovery, viability, as well as phenotype expression after cryopreservation of immature DC was considered in this study. DC were induced to maturation and evaluated in FACS analysis for phenotype expression and proliferation assays. Mature DC were able to generate an allogeneic T-cell response as well as an anti-CMV response as detected by proliferation assays. These data indicate that the described large-scale GMP-compatible system results in the generation of stable DC derived from one volume of blood processed, which are qualitatively and quantitatively sufficient for clinical application in immunotherapeutic protocols.

Transfer of the Multidrug Resistance Gene, MDR-1, to Hematopoietic Progenitors from a Patient with Transformed Large Cell Lymphoma, and Demonstration of In Vivo Chemotherapy Protection with Dose Escalating Chemotherapy.

We report results for the first patient enrolled in a gene therapy clinical trial (RAC and FDA approved) for poor risk non-Hodgkin's lymphoma. The retroviral vector, SF1m (Hildinger et al '98,'99), consists of the LTRs of SFFV, the leader sequence of MESV, and the full length MDR-1 cDNA. The first subject enrolled on this trial had follicular transformed large cell lymphoma that relapsed with a large mediastinal mass and superior vena cava syndrome two and a half years after initial therapy. After 3 cycles of salvage chemotherapy with RICE, a residual 4 cm mediastinal mass persisted that was gallium avid, and B symptoms recurred by 3 weeks. With informed consent, the patient underwent mobilization with rituximab, cyclophosphamide, and G-CSF. A total of 3 X 106 CD34+ cells/kg were cryopreserved without further manipulation. An additional apheresis product was subjected to CD34 selection on the Isolex 300i. The purified product was 96% CD34+, represented 3.17 X 106 CD34+ cells/kg, 98% yield with 99% viability. These cells were pre-incubated for 17h with 20 ng/ml IL-3, and 100 ng/ml each of IL-6, SCF and TPO, and then transduced with three changes of retrovirus SF1m in fibronectin CH-296 peptide (Retronectin)-coated gas permeable bags (X-fold), alternately 7–9 hours with virus, followed by 6–11 hours in media with cytokines, and a final centrifugation. The final product was 95% CD34+, with 96% viability, and represented 5.5 X 106 CD34+ cells/kg, because of a 2.4-fold expansion. Post transduction, 10% of the cells effluxed rhodamine (a function of the MDR-1 encoded p-glycoprotein), as compared to 1% of the non-transduced cells. The patient received low dose (100 cGy) total body irradiation (TBI), followed by infusion of the transduced cells. The patient then received 4 cycles of vincristine, dose-escalating etoposide, and prednisone, and the platelet count nadir improved with each cycle (18, 65, 98, and 131K after cycles 1, 2, 3, and 4), as did the ANC nadir by the final cycle (100 cycle 1 v. 640 cycle 4). The patient had no evidence of disease prior to a second, myeloablative transplant using the original unmanipulated cells infused after high dose cyclophosphamide and TBI (1200 cGy). The grade 4 adverse events included febrile neutropenia and thrombocytopenia after the first cycle of dose escalating chemotherapy, and after the myeloablative transplant. At one year after infusion of transduced cells, the patient remains well and free of disease. Thus, a patient who was poor risk for autologous transplant initially, due to rapid re-growth of disease, was brought to a myeloablative transplant without residual disease by this treatment with gene modified cells. The striking improvement in nadir platelet count was presumably due to expansion of MDR-1 expressing cells. Lastly, the final, fully myeloablative transplant should represent a safety feature, protecting against the development of a malignancy due to insertional mutagenesis, as the gene modified cells would not be expected to survive.

Impact of Leukapheresis Cell Composition on Immunomagnetic Cell Selection with the Baxter Isolex 300i Device: A Statistical Analysis

Immunomagnetic cell selection (ICS) of CD34(+) cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34(+) ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34(+) cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34(+) cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34(+) cells (>500 x 10(6)) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

Impact of Leukapheresis Cell Composition on Immunomagnetic Cell Selection with the Baxter Isolex 300i Device: A Statistical Analysis

Immunomagnetic cell selection (ICS) of CD34+ cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34+ ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34+ cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34+ cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34+ cells (<500 × 106) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

A protocol for generation of clinical grade mRNA-transfected monocyte-derived dendritic cells for cancer vaccines.

With the aim of producing large quantities of mRNA-transfected monocyte-derived dendritic cells (DCs) to be used as cancer vaccines, a new clinical grade procedure has been developed. Peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis were enriched for monocytes by immunomagnetic depletion of CD19+ B cells and CD2+ T cells employing the ISOLEX 300i device. After 5 days of culture of enriched monocytes in gas permeable Teflon bags, using serum-free medium supplemented with granulocyte/macrophage-colony stimulating factor and interleukin-4 (IL-4), immature DCs were generated. Following transfection with mRNA from three human prostate cancer cell lines (DU145, LNCaP and PC-3), employing a newly developed square wave electroporation procedure, the immature DCs were immediately transferred to Teflon bags and matured for 48 h, using serum-free medium supplemented with IL-1alpha, IL-6, tumour necrosis factor-alpha and PGE2. The electroporation procedure efficiently transferred mRNA into the DCs with minor effect on the viability of the cells. The generated matured transfected DCs show high expression of the antigens CD83, CD80, CD86 and human leucocyte antigen-DR. Freezing and thawing of the transfected matured DCs had minor effect on cell viability and the phenotype. From 4 x 109 PBMCs, about 1 x 108 transfected matured DCs are produced. The thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by enzyme-linked immunospot assay using mock-transfected DCs as control. Based on these results, clinical trials in cancer patients have been initiated.

Factors affecting purification of CD34(+) peripheral blood stem cells using the Baxter Isolex 300i.

A total of 201 patients with breast cancer, ovarian cancer, or hematological malignancies underwent mobilization of peripheral blood stem cells (PBSC) using chemotherapy and granulocyte-colony stimulating factor (G-CSF). Stem cell products were collected using the Baxter CS3000 pheresis machine. The Baxter Isolex 300i was used to perform 240 CD34(+) cell separations on the apheresis products. Factors affecting yield and purity of the CD34(+) cells were analyzed. Overall yield was 55% and overall purity was 91.7%. T cell contamination was limited to 0.43% of total cells. Variables including red blood cells (RBC) concentration, platelet concentration, CD34(+) cell concentration, total WBCs selected, and time until processing had little effect on yields and purities. Installation of version 2.5 of the software in the Isolex 300i showed a modest improvement in yield and purity. Patients were reinfused with the cryopreserved CD34(+) selected cells following high-dose chemotherapy. No infusion-related side effects were noted. Analysis of engraftment data using the CD34(+)-selected cells revealed an increased risk of delayed or failed platelet engraftment when <5.0 x 10(6) CD34(+) cells per kilogram were transplanted. The Baxter Isolex 300i provides reproducible CD34(+) cell purification over a wide range of starting conditions. To provide prompt engraftment, >5.0 x 10(6) CD34(+) cells per kilogram should be infused for transplantation.

Clinical-scale selection of anti-CD3/CD28-activated T cells after transduction with a retroviral vector expressing herpes simplex virus thymidine kinase and truncated nerve growth factor receptor.

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.

High-dose chemotherapy and CD34-selected peripheral blood progenitor cell transplantation for patients with breast cancer metastatic to bone and/or bone marrow.

Fifty women with breast cancer metastatic to bone or bone marrow involvement on light microscopy at the time of initial evaluation were treated with high-dose chemotherapy (HDC) and peripheral blood progenitor cell (PBPC) transplantation with CD34(+) cell selection using the Isolex 300i system. All patients received induction chemotherapy. PBPC were mobilized with chemotherapy and granulocyte colony-stimulating factor. The median CD34(+) progenitor purity was 94.7% (range 72-98.7%) and recovery 38.4% (range 21-60%). Forty-eight hours after HDC with cyclophosphamide, cisplatin and carmustine, PBPC were reinfused. Median time to neutrophil count >0.5 x 10(9)/l was 9 (range 9-12) days and to platelet transfusion independence 11 (4-30) days. These data demonstrate that selected CD34(+) PBPCs allow rapid hematologic reconstitution after HDC. During follow-up, 23% of patients developed herpes zoster. Two patients developed cytomegalovirus infections. Three patients developed fungal infections. The development of these infections was not associated with steroid use but appeared more frequently in patients with diabetes mellitus. Seventy-four per cent of patients received steroids for pulmonary toxicity. Treatment-related mortality was 4%. Progression-free survival and overall survival at 35 months was 22.4% and 40.5%, with a median of 11.4 months and 15.4 months, respectively.

CD34 selection using three immunoselection devices: comparison of T-cell depleted allografts

T-cell depletion of allografts markedly reduces the incidence of GvHD following BMT. The approach taken at our Center has utilized the physical separation method of counterflow centrifugal elutriation (CCE), augmented by recovery of stem cells from lymphocyte-rich fractions by immunoaffinity selection of CD34(+) stem cells. We wanted to compare the performance characteristics of three commercially available selection devices, as well as the clinical outcomes of patients who received allografts engineered by the different devices. BM allografts were prepared for patients undergoing BMT for hematologic malignancies. BM cells were separated into lymphocyte-rich and lymphocyte-depleted fractions using CCE, followed by recovery of CD34(+) cells from the lymphocyte-rich fraction using one of three immunoselection devices [CellPro CEPRATE, Nexell Isolex 300i (software version 2.5) and AmCell CliniMACS]. Allografts consisted of the lymphocyte-depleted fraction plus the CD34-selected fraction. Yields of CD34(+) cells were comparable for the three devices. However, there were significant differences in purity (CEPRATE < Isolex 300i < CliniMACS) and time from start of fractionation to infusion (CEPRATE < CliniMACS < Isolex 300i). More technical problems were encountered with the Isolex 300i device. Allograft compositions were comparable. Transplant outcomes (engraftment and incidence of GvHD) also were comparable. Qualitatively and quantitatively, allografts prepared with the CEPRATE, Isolex 300i (v 2.5) and CliniMACS devices should be considered comparable for use in this setting and probably also for direct T-cell depletion of BM.

Comparison of monocyte enrichment by immuno-magnetic depletion or adherence for the clinical-scale generation of DC

DC generated from monocytes have been used for vaccines. We have developed a monocyte enrichment procedure by depleting T and B cells with anti-CD2 and anti-CD19 Abs using the automated Isolex 300i magnetic cell selector for clinical-scale DC generation in gas permeable SteriCell culture bags. We have also compared DC function, yield and purity of DC generated from adherent monocytes using culture bags in a closed system, with DC generated in conventional tissue culture flasks. Monocytes were enriched from normal donor apheresis products using CD2/19 depletion with experimental software on the Isolex 300i (ISO), adherence (AD) to SteriCell bags and to T175 flasks and then cultured for 7 days in serum-free X-VIVO 15 media with GM-CSF and IL-4. Phenotype and dextran uptake were analyzed by flow cytometry and allogeneic MLR was also evaluated. ISO-DC and AD-DC from SteriCell bags showed similar viability. Higher purity of ISO-DC than AD-DC was measured by forward- and side-scatter flow cytometry. Similar expression of CD1a, CD80, CD86 and CD83 were observed in both ISO-DC and AD-DC. Similar dextran uptake and allo MLR were also observed. These data indicated that functional DC were generated in gas permeable SteriCell culture bags from both ISO- and AD-monocytes in a closed system.