Peripheral Blood Leukapheresis Research Articles

Choosing T-cell sources determines CAR-T cell activity in neuroblastoma.

The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.

Predicting the CD34 content of mobilized peripheral blood leukapheresis products: single institution experience over 20 years

Background aimsSince the standardization of CD34 measurement by flow cytometry, predictors of leukapheresis CD34 yield have played a pivotal role in planning donor leukaphereses. We describe here a single institution's experience with a multivariate predictor that was used for 2,929 products without alteration for 20 years. MethodsThe ordinary least squares regression model variables included log peripheral CD34 count, collection duration (3- versus 4-hours), collection number, donor sex, and transplant type. ResultsDuring the study period we changed flow cytometers twice and leukapheresis instruments once. During the Cobe Spectra era the predictor explained 90% of the variability in CD34 collection yield for autologous transplants (r2 = 0.90), and 70% for allogeneic transplants with an overall sensitivity to predict a CD34 yield of ≥ 1 × 106/kg of 97.7%, and specificity of 81.4%. ConclusionsImplemented prospectively with real-time result reporting, the model allowed us to predict CD34 yield with both 3- and 4-hour collection scenarios. Given this guidance, 3-hour collections were selected by the clinical team 25% of the time, saving patient leukapheresis time and resources. When faced with a prediction of < 1 × 106 CD34/kg, the clinical team chose to defer collection 72% of the time. In instances where leukapheresis was performed despite a poor predicted outcome, 85% of patients collected on the Cobe Spectra, and 92% of patients collected on the Optia, failed to collect at least 1 × 106 CD34/kg. A revised model is tested retrospectively on Optia data, and suggestions for further improvements are discussed.

POINT‐OF‐CARE ANTI‐BCMA CAR T‐CELL THERAPY INDUCES ENCOURAGING RESPONSE RATES IN HIGH‐RISK RELAPSED/REFRACTORY MULTIPLE MYELOMA

Introduction: Anti-BCMA chimeric antigen receptor (CAR) T-cell therapy showed excellent efficacy in patients with relapsed/refractory multiple myeloma (R/R MM). Point-of-care (POC) CAR manufacturing abrogates the need for cryopreservation and shipment of cells, thus shortening the manufacturing process and reducing the necessity of bridging therapy. We report outcomes of phase 1b/2 single-center clinical trial of autologous POC anti-BCMA CAR T-cell therapy in patients (pts) with R/R MM treated with ≥3 prior therapies (NCT05243212). Methods: Pts underwent a single peripheral blood leukapheresis. Fresh T-cells were transduced with retroviral vector encoding the anti-BCMA CAR (based on 11D5-3 ScFv, CD28 costimulatory domain, and CD3-ζ signaling domain). Cell dose was 6 × 106/kg (dose level I [n = 3]) or 9 × 106/kg (dose level II [n = 29]) CAR T-cells. Response was defined per IMWG criteria. Last follow-up was as of 02/2023. Results: All 32 pts (median age 60, IQR 54–67) enrolled received CAR T-cell infusion in a median of 11 days (IQR 11–11) after leukapheresis. Only 2 pts received bridging chemotherapy. The median number of prior therapies was 4 (IQR 3–5), with 59% and 34% of the pts being penta- and quad-refractory, respectively. 7 (22%) pts had prior exposure to BCMA-targeted therapy (belantamab mafodotin, n = 5 [16%]; talqeutamab, n = 2 [6%]). At enrollment, 10 (34%) pts had high-risk cytogenetics, 7 (22%) had double-hit myeloma, and 17 (57%) had extramedullary involvement. Only 17 (53%) and 2 (16%) pts were eligible to enroll in the KarMMa (NEJM, 2021) and CARTITUDE-1 (Lancet, 2021) studies, respectively. One patient (3%) developed grade 3 cytokine release syndrome. Immune effector cell-associated neurotoxicity syndrome was not noted. Grade 3–4 neutropenia and thrombocytopenia occurred in 31 (97%) and 16 (50%), respectively. Anemia requiring transfusion occurred in 14 (44%) pts. Cellular therapy-related death was not observed. The median follow-up was 3.9 months (IQR 2.6–7.3). Best Overall response rate (PR at least) was 59% (VGPR at least, 40%). Median time to first response was 31 days (95% CI: 26–33). Estimated 6-months OS, PFS and duration of response were 89% (95% CI: 75–100), 48% (95% CI: 33–72), and 63% (95% CI: 41–97), respectively. Patients with prior exposure to BCMA-targeted therapies had an inferior PFS (HR 3.4 [95% CI: 1.2–9.7] p = 0.03; Figure 1). Conclusion: POC anti-BCMA CAR T-cells induced high response rates with an excellent safety profile in high-risk MM patients mostly not eligible to be enrolled in the pivotal trials. The rapid CAR-T production time obviated the need for bridging therapy in most patients. It is noteworthy that prior exposure to BCMA-targeted therapies is associated with dismal PFS, hence those therapies should be carefully considered when CAR T-cell therapy might be intended. Encore Abstract—previously submitted to EHA 2023 Keywords: cellular therapies, multiple myeloma No conflicts of interests pertinent to the abstract. * H. Magen, E. Shkury and A. Avigdor contributed equally to the abstract.

Naïve T Cell-Depleted Hematopoietic Graft Combined with Solid Organ Transplantation As Strategy to Induce Tolerance By Transient Mixed Chimerism

BACKGROUND The major challenge of solid organ transplantation (SOT) is to prevent graft rejection. The chronic use of pharmacological immunosuppression has been associated with increased rates of infection, cardiovascular, metabolic, endocrine disorders, and cancer. The induction of chimerism by donor hematopoietic stem cell transplantation (HSCT) together with SOT is an active tolerance induction approach. Here, we present a new model for inducing transient mixed chimerism in 2 patients undergoing SOT employing a non-myeloablative conditioning (NMA) regimen and a partially T cell-depleted (TCD) graft consisting of CD34+ hematopoietic progenitor cells and CD45RA− memory lymphocytes. METHODS SOT and HSCT were performed at University Hospital La Paz, Madrid. Bone marrow harvesting was performed on the iliac crest and lumbar and dorsal vertebrae of the deceased donor for patient#1 and mobilized leukoapheresis from sibling donor for patient#2. First we obtained the enriched by CD34+ selection using the CliniMACS system (Miltenyi Biotec) and afterwards the CD45RA− cells were purified. Lineage-specific chimerism analysis was analyzed using quantitative real-time polymerase chain reaction. Immune cell recovery was monitored by multiparametric flow cytometry. We obtained the permission of the Organización Nacional de Trasplantes and the informed consent was obtained from the patient and donor and/or caregivers. RESULTSPatient 1 A 15-year-old boy was diagnosed with intestinal epithelial dysplasia in 2008. After two previous intestinal and multivisceral acute cellular rejections forced a third MVT. With the imptemption to induce donor tolerance, a T cell-depleted bone marrow graft from the same deceased donor was employed. Bone marrow harvesting was performed obtaining a total volume of 141 ml with 4x109 total nucleated cells. Peripheral blood was obtained after unclasping the thoracic aorta. The stem cell fraction from bone marrow was enriched by CD34+ selection, obtaining 8.75x105/kg CD34+cells/kg. Memory lymphocytes were purified and aliquots containing 1x106/kg CD3+CD45RO+ and 2.1x104/kg CD3+CD45RA+ T cells. Both products were cryopreserved. The conditioning regimen before HSCT included fludarabine and cyclophosphamide. On day 88 after MVT, we infused all the CD34+ cells and 1x106/Kg CD3+CD45RO+ T cells. The CD3+CD45RO+ T cells were also infused periodically on a monthly basis for 1 year. Donor chimerism in CD3+ T cells ranged from 0.05 to 5.1 during the first year after HSCT. We observed a peak of the percentage of regulatory T cells (Tregs). No degree of graft-versus-host disease (GvHD) was observed, and the patient became independent of total parenteral and enteral nutrition. Serial follow-up intestinal biopsies did not show any sign of immune rejection. The patient continued to receive sirolimus to maintain drug concentrations at 6 ng/ml as the only immunosuppression treatment. No signs of rejection were detected after 27 months of follow-up. Patient 2 A 49-year-old woman was diagnosed with chronic kidney failure secondary to rapidly progressive glomerulonephritis. The patient had received 3 previous kidney transplants from deceased donors. A kidney transplant from her sibling was performed in March 2021. Hematopoietic progenitor cells were obtained from mobilized peripheral blood leukapheresis. A NMA conditioning regimen employing total lymphoid irradiation (8 Gy) with antithymocyte globulin (7.5 mg/kg). Two weeks after the kidney transplantation, we infused a graft consisting of 1.27x107/kg CD34+, 4.43x106/kg CD3+CD45RO+, and 5x104/kg CD3+CD45RA+ cells. Controls for hematopoietic chimerism demonstrated the presence of transient mixed hematopoietic chimerism during the first 2 months after SOT of between 0.1% and 40% CD3+ cells. The patient continued on monotherapy with tacrolimus. Immune reconstitution analysis showed a Treg peak. The patient did not have acute rejection, GvHD, viral reactivations or episodes of bacterial infection during the 17 months of follow-up. CONCLUSIONS We describe for the first time the use of non-myeloablative conditioning and a CD45RA− T cell-depleted graft to induce transitory mixed chimerism using both hematopoietic progenitors and a solid organ coming from the same donor, for tolerance induction purposes. The patients continue to progress favorably 17 months or more after SOT, receiving immunosuppressive monotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 1069: Autologous γδ T cell therapy for treatment-refractory non-small-cell lung cancer: An open-label, single-arm, multicenter, phase II study

Abstract Background: Only a fraction of non-small cell lung cancer (NSCLC) patients possess targetable driver mutations, and the response rates of immune checkpoint blockades are still unsatisfactory. Therefore, the development of more effective therapies is needed for treatment-refractory NSCLC. We conducted a phase I clinical study of autologous γδ T cell therapy for NSCLC patients and reported safety and feasibility. Here, we report the result of phase II clinical trial of adoptive γδ T cell transfer therapy for treatment-refractory NSCLC patients. Methods: Patients with NSCLC who had undergone at least two regimens of standard chemotherapy for unresectable disease or who had undergone at least one treatment including chemotherapy or radiation for recurrent disease after surgery were enrolled in this open-label, single-arm, multicenter, phase II study. After preliminary testing of γδ T cell proliferative capacity, mononuclear cells of the patient collected by peripheral blood leukapheresis were cultured with zoledronic acid and IL-2. Expanded autologous γδ T cells (&amp;gt;1 × 109) were transferred intravenously every two weeks for a total of six injections. If the patient perceived some clinical benefit, the patient could continue further treatments until the disease became progressive. The primary endpoint of this study was progression-free survival (PFS), and the secondary endpoints included overall survival (OS), best objective response rate (ORR), disease control rate (DCR), and safety. The clinical efficacy of the treatment was determined if the median PFS is significantly longer than 3 months. Results: Autologous γδ T cell therapy was given for 25 patients, and 16 patients completed the 6 courses of the treatment. Among the 25 patients, 20 had adenocarcinoma, 4 squamous cell carcinoma and 1 large cell carcinoma. The median PFS was 95.0 days (95% CI 73.0-132.0 days). The median OS was 418.0 days (179.0-479.0 days). The best overall responses were 1 partial response (PR), 16 stable disease (SD) including 4 patients whose time to progression was longer than 180 days, and 8 progressive disease (PD). ORR and DCR were 4.0% (0.1-20.4%) and 68.0% (46.5-85.1%), respectively. Severe adverse events (SAE) were developed in 9 patients during the study, including pleural effusion, anorexia, cough, dyspnea, respiratory failure, pneumonitis, ascites, tumor pain, and intracranial hemorrhage. Most of them were associated with disease progression; however, a case of pneumonitis was related to γδ T cell therapy. Conclusion: Although the trial did not meet its primary efficacy endpoint, the results of this study indicate that autologous γδ T cell therapy was well tolerated and may have an acceptable disease control rate. Considering the unique mechanism of action towards tumor cells, γδ T cell therapy may be a candidate counterpart of combination therapy for treatment-refractory NSCLC. Citation Format: Kazuhiro Kakimi, Hirokazu Matsushita, Takamichi Izumi, Keita Masuzawa, Takahiro Karasaki, Shinnosuke Ikemura, Kentaro Kitano, Ichiro Kawata, Tadashi Manabe, Tomohiro Takehara, Toshiaki Ebisudani, Kazuiro Nagayama, Hiroyuki Yasuda, Masaaki Sato, Kenzo Soejima, Jun Nakajima. Autologous γδ T cell therapy for treatment-refractory non-small-cell lung cancer: An open-label, single-arm, multicenter, phase II study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1069.

Manufacture of endothelial colony‐forming progenitor cells from steady‐state peripheral blood leukapheresis using pooled human platelet lysate

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44×10-8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730×103 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP.

Myasthenia Gravis Treated With Autologous Hematopoietic Stem Cell Transplantation

Some patients with myasthenia gravis (MG) do not respond to conventional treatment and have severe or life-threatening symptoms. Alternate and emerging therapies have not yet proved consistently or durably effective. Autologous hematopoietic stem cell transplant (HSCT) has been effective in treating other severe autoimmune neurologic conditions and may have similar application in MG. To report 7 cases of severe MG treated with autologous HSCT in which consistent, durable, symptom-free, and treatment-free remission was achieved. This retrospective cohort study reports outcomes at The Ottawa Hospital, a large, Canadian, tertiary care referral center with expertise in neurology and HSCT, from January 1, 2001, through December 31, 2014, with a median follow-up of 40 months (range, 29-149 months). Data collection and analysis were performed from February 1 through August 31, 2015. All patients with MG treated with autologous HSCT at The Ottawa Hospital were included. All had persistent severe or life-threatening MG-related symptoms despite continued use of intensive immunosuppressive therapies. Autologous hematopoietic stem cell grafts were mobilized with cyclophosphamide and granulocyte colony-stimulating factor, collected by peripheral blood leukapheresis, and purified away from contaminating lymphocytes using CD34 immunomagnetic selection. Patients were treated with intensive conditioning chemotherapy regimens to destroy the autoreactive immune system followed by graft reinfusion for blood and immune reconstitution. The primary outcome was MG disease activity after autologous HSCT measured by frequency of emergency department visits and hospitalizations and Myasthenia Gravis Foundation of America (MGFA) clinical classification, MGFA therapy status, and MGFA postintervention status. Safety outcomes included all severe autologous HSCT-related complications. Seven patients underwent autologous HSCT, 6 for MG and 1 for follicular lymphoma with coincident active MG. Mean (SD) ages at MG diagnosis and at autologous HSCT were 37 (11) and 44 (10) years, respectively. Five patients (71%) had concurrent autoimmune or lymphoproliferative illnesses related to immune dysregulation. All patients had distinct clinical and electromyographic evidence of MG (MGFA clinical classification IIIb-V). All patients achieved durable MGFA complete stable remission with no residual MG symptoms and freedom from any ongoing MG therapy (MGFA postintervention status of complete stable remission). Three patients (43%) experienced transient viral reactivations, and 1 (14%) developed a secondary autoimmune disease after autologous HSCT, all of which resolved or stabilized with treatment. There were no treatment- or MG-related deaths. Autologous HSCT results in long-term symptom- and treatment-free remission in patients with severe MG. The application of autologous HSCT for this and other autoimmune neurologic conditions warrants prospective study.

Peripheral stem cell collection: From leukocyte growth factor to removal of catheter

A wide variety of hematologic malignancies, autoimmune diseases, inborn errors of metabolism, and bone marrow failure syndromes maybe put into a remission or potentially cured by hematopoietic stem cell transplantation (HSCT). Depending on the underlying disorder, stem cells may be collected from the future transplant recipient themselves, an human leukocyte antigen (HLA) identical donor, a mismatched related or unrelated donor, or banked umbilical cord blood. Peripheral blood leukapheresis to collect stem cells from autologous or allogeneic donors is safe and well-tolerated. In most cases, the apheresis procedure is performed in an outpatient setting over 4-6 h a day for up to 4 days to achieve the stem cell dose needed for HSCT. Current research in stem cell mobilization and collection is focused on optimizing the numbers of stem cells collected by improving mobilization regimens, and increasing the efficiency of the apheresis collection to reduce the number of apheresis procedures needed to meet the stem cell dose requirement for HSCT.

Stem cell mobilization and harvesting by leukapheresis alters systemic cytokine levels in patients with multiple myeloma

Background aimsStem cell mobilization and harvesting by peripheral blood leukapheresis in patients with myeloma can alter plasma levels of certain cytokines. In the present study, we investigated the effects of these interventions on a larger group of cytokines. MethodsPlasma cytokine levels were determined in 15 patients with myeloma who were undergoing peripheral blood stem cell harvesting, and we compared the patients with healthy donors who were undergoing platelet apheresis. ResultsSeveral cytokines showed altered levels in patients with myeloma when examined after chemotherapy plus granulocyte colony-stimulating factor–induced stem cell mobilization. The most striking effect was increased levels of several CCL (CCL2/3/4) and CXCL (CXCL5/8/10/11) chemokines as well as increased thrombopoietin, interleukin 1 receptor antagonist, interleukin-4, granulocyte colony-stimulating factor and hepatocyte growth factor. Stem cell harvesting by apheresis altered the plasma levels of several mediators (CD40 ligand, interleukin 1 receptor antagonist, CCL5 and CXCL5/8/10/11). Apheresis in patients with myeloma had divergent effects on these chemokine levels, although they were all still significantly higher than for healthy individuals. Thrombapheresis in healthy individuals had only minor effects on plasma cytokine levels. Stem cell graft supernatants showed high levels of several cytokines, especially CCL and CXCL chemokines. Analyses of chemokine profiles in pre-apheresis plasma and graft supernatants suggested that such profiling can be used to detect prognostically relevant differences between patients. ConclusionsOur results demonstrate that patients with myeloma have an altered cytokine network during stem cell mobilization, and the network is further altered during stem cell harvesting by leukapheresis. These treatment- or procedure-induced alterations involve several mediators known to affect myeloma cell proliferation, migration and survival.

Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro

Low-intensity pulsed ultrasound (LIPUS) stimulated the viability, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) from fresh and cryopreserved peripheral blood leukapheresis product, as well as cord blood when applied for 10 min each day for 4 days. Cell viability, proliferation and differentiation were assessed on day 5 by viable cell counting, MTS proliferation assay, flow cytometry, and colony-forming unit assay. LIPUS stimulation: (i) enhanced the proliferation of fresh HSPC and maintained the viability of cryopreserved HSPC in vitro; (ii) did not affect the percentage of CD34(+) and CD14(+) cells; and (iii) enhanced burst-forming unit-erythroid colony formation. Hence, we suggest that this novel LIPUS stimulation approach might enhance the efficacy of clinical transplantation and cellular therapies using HSPC.

A Highly Efficient, Clinically Applicable Transfection Method to Redirect the Specificity of Immune Cells and Enhance Their Anti-Tumor Capacity

Effective immunotherapies rely on triggering specific cytotoxic responses against target cells. Chimeric Antigen Receptors (CAR) composed of antigen recognition domains in the form of scFv portions of monoclonal antibodies and immune cell activation domains provide non-MHC restricted, KIR-independent, immune cellmediated cytotoxicity directed toward target cells expressing specific antigens. It is well demonstrated that CAR transfection into immune cells using viral vectors redirects their specificity and enhances their cytotoxicity. Translation of these findings into clinically feasible treatment strategies is challenged by the labor/costs associated with extended cell processing, and by safety concerns associated with viral vectors. We describe here the use of non-viral methods to deliver a nucleotide sequence encoding CAR into freshly isolated immune cells obtained from peripheral blood leukapheresis collection. Protocols for loading mRNA encoding eGFP (irrelevant control) and a previously described anti-CD19 CAR (Imai et al. Blood 2005) into peripheral blood mononuclear cells (PBMC) and cell subsets (T cells, NK cells, and monocyte-depleted PBMC) were developed and optimized using a clinically validated, automated, closed-system, flow electroporation technology. PBMC were obtained from 5 normal healthy donors and 1 patient with B-cell chronic lymphocytic leukemia (B-CLL). Expression levels of eGFP or anti-CD19-BB-z were analyzed by flow cytometry. Specific cytotoxicity of target cells after 4 hours was measured by flow cytometry using acetoxymethyl-calcein labeled CD19+ target cells which included acute lymphoblastic leukemia (ALL) cell lines, primary B-CLL cells and autologous B cells. Following loading of mRNA encoding eGFP, cell viability was consistently ≥80% for all cell populations. Most cells expressed eGFP: 97±1% (day 1) and 83±2% (day 7). Following loading of mRNA encoding anti-CD19-BB-z, cell viability was 96%±1% for resting T cells (n=2), 93%±10% for PBL (n=2), 94%±8% for PBMC (n=3), 80%±15% for NK cells (n=4). Expression of anti-CD19-BB-z protein receptor was 54%±11% (day 1) in PBMC (n=6). CAR expression decreased over time and was essentially similar to baseline on day 7 post-loading. Cells expressing anti-CD19-BB-z protein (but not those expressing eGFP only), lysed 60%–80% ALL or B-CLL target cells at an effector-to-target ratio (E:T) of 8:1. A shigher E:T ratio of 20:1 was required for 60%–80% lysis of autologous B-cell targets. There was no change in cytolytic activity of CAR mRNA loaded-PBMC during the first 3 days post-loading. Subsequently, cytolytic activity dropped and on day 7 (post-loading) was approximately half of the activity measured on day 3 (post-loading).In vivo testing was performed in a sub-cutaneous tumor grafting model in Beige SCID mice. The mice (N = 5 per group) were transplanted subcutaneously with CD19+ tumor (HS Sultan) cells (1 × 106) co-mixed with PBMC loaded with anti-CD19-BB-z mRNA (doses = 0.74, 2.22, 6.67 and 20 ×106). Controls were tumor cells only or tumor cells co-mixed with unmodified PBMC. Tumors developed and grew in 3/5 animals in each control group and in 2/5 in lowest anti-CD19-BB-z mRNA-loaded dose. No tumor formation was observed in any animal in each of the 3 higher cell doses. More comprehensive animal studies and investigation of engineering additional constructs for chimeric signal receptor with tumor affinities other than anti-CD19 are in progress. These results indicate that CAR can be effectively transfected into immune cells with the methodology that we developed. Because of its simplicity and effectiveness, this methodology is well suited for clinical application and holds promise for improving the specificity and widening the feasibility of cell therapy.

Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient

AbstractWe found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte–colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)– and bone marrow (BM)–derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.

Expression of a functional eotaxin (CC chemokine ligand 11) receptor CCR3 by human dendritic cells.

Critical to the function of Ag-presenting dendritic cells (DCs) is their capacity to migrate to lymphoid organs and to sites of inflammation. A final stage of development, termed maturation, yields DCs that are strong stimulators of T cell-mediated immunity and is associated with a remodeling of the cell surface that includes a change in the levels of expression of many molecules, including chemokine receptors. We show in this study that CCR3, a chemokine receptor initially discovered on eosinophils, is also expressed by human DCs that differentiate from blood monocytes, DCs that emigrate from skin (epidermal and dermal DCs), and DCs derived from CD34+ hemopoietic precursors in bone marrow, umbilical cord blood, and cytokine-elicited peripheral blood leukapheresis. Unlike other chemokine receptors, such as CCR5 and CCR7, the expression of CCR3 is not dependent on the state of maturation. All DC subsets contain a large intracellular pool of CCR3. The surface expression of CCR3 is not modulated following uptake of particulate substances such as zymosan or latex beads. CCR3 mediates in vitro chemotactic responses to the known ligands, eotaxin and eotaxin-2, because the DC response to these chemokines is inhibited by CCR3-specific mAbs. We postulate that expression of CCR3 may underlie situations where both DCs and eosinophils accumulate in vivo, such as the lesions of patients with Langerhans cell granulomatosis.

High-Efficiency Gene Transfer Into Ex Vivo Expanded Human Hematopoietic Progenitors and Precursor Cells by Adenovirus Vectors

Replication-deficient adenoviral vectors (AdVec), which infect cycling and noncycling cells with high efficiency, low toxicity, and ease of delivery, provide ideal vehicles to study the expression of regulatory genes controlling different stages of hematopoiesis. To examine the infection efficiency of AdVec in hematopoietic precursor and progenitor cells, we used a replication-deficient adenovector expressing the humanized form of the cDNA for green fluorescent protein (AdGFP), permitting assessment of infection efficiency and kinetics of transgene expression in viable hematopoietic cells using flow cytometry and fluorescence microscopy. Flow-cytometric analysis of ex vivo expanded hematopoietic precursor cells infected with a multiplicity of infection (MOI) of 100 of AdGFP show that 78% of megakaryocytic (CD41a+ and CD42b+) cells, 82% of dendritic (CD1a+) cells, 41% of RBC precursors (glycophorin A+), and 32% of monocytic (CD14(+)) cells expressed GFP. Nineteen percent +/- 1% of freshly isolated CD34(+) cells from peripheral blood leukapheresis products infected under the same conditions expressed GFP. Morphologic evaluation of ex vivo expanded, AdGFP-infected CD34(+) cells showed normal maturation. The functional capacity of AdGFP-infected CD34(+) cells was analyzed by quantifying clonogeneic efficiency and proliferative capacity. Infection of CD34(+) progenitor cells with MOIs of 1 to 100 did not impair clonogeneic efficiency of CD34(+ )cells. However, MOI greater than 100 resulted in a significant inhibition of colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) formation. In sequential dilution expansion over 3 weeks (Delta assay), the cytokine-driven proliferative potential of CD34(+) cells was not impaired following exposure to AdGFP at MOIs of 1 to 1,000. The GFP+ population expanded 10- to 15-fold at high MOIs (500 to 1,000), indicating multiple copies of the transgene in the initially infected CD34(+) cells, which were expressed in subsequent progenies. These data show that AdVec deliver transgenes with high efficiency and low toxicity to hematopoietic progenitor and precursor cells. Introduction of marker genes such as GFP into hematopoietic cells by AdVec will provide a valuable system for study of development, homing, and trafficking of hematopoietic precursor and progenitor cells in vitro and in vivo. Furthermore, these results provide insights into the design of gene therapy strategies for treatment of hematologic disorders by AdVec.

CD34+ cell engraftment, ex vivo expansion, and malignant cell depletion following immunomagnetic selection.

This review describes the published preclinical and clinical data on the use of a manual or semiautomated immunomagnetic selection device, termed the Isolex system. Preclinical evaluation of hematopoietic progenitor cells (CD34+ cells) selected from bone marrow, peripheral blood leukapheresis products, and umbilical cord blood is reviewed with respect to differentiation (CFU-GM, BFU-E, and CFU-GEMM formation) and proliferation. The purities and yields of CD34+ cell products from clinical trials performed since 1994 are presented along with data on malignant cell depletion. On average, the Isolex system resulted in a final product median purity of 67% and a final product median yield of 64%. Positive selection of CD34+ cells with this device decreased residual tumor cell levels by 2-3 logs in autologous transplant products and reduced T cell levels by 3-4 logs in allogeneic grafts. To evaluate the clinical effect of these immunomagnetically selected cells, data on the rate of engraftment were reviewed. Autologous CD34+ cell transplantation resulted in recovery time from neutropenia (ANC > 500/microliter) of 9-14 days and recovery time from thrombocytopenia (platelet count > 20,000/microliter) of 10-20 days. These data showed that the Isolex system can positively select progenitor cells to reconstitute the hematopoietic system following myeloablative therapy.

Comparison of the content and subpopulations of CD3 and CD34 positive cells in bone marrow harvests and G-CSF-mobilized peripheral blood leukapheresis products from healthy adult donors

Recombinant human granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem/progenitor cells (PBPC) have replaced bone marrow (BM) harvests for autologous transplantation after myeloablative therapy in cancer patients. G-CSF-mobilized PBPC from healthy donors contain one log excess of T lymphocytes representing a potential risk for graft-versus-host disease (GVHD). However, recent pilot clinical studies of G-CSF-mobilized allogeneic PBPC transplantation have shown rapid haematological recovery and no severe acute GVHD except in a very few cases. Therefore, the risk of inducing severe acute GVHD is not as high as was expected during the pioneering period of allogeneic PBPC transplantation. The present study was performed to address the possible reasons for the rapid haematological recovery and the absence of severe acute GVHD observed after allogeneic PBPC transplantation by comparing the contents and subsets of CD3 + and CD34 + G-CSF-mobilized PBPC ( n = 31) with those of BM ( n = 26) allografts from healthy adult donors. The present results revealed that the phenotypic profiles of CD3 + and CD34 + cells differ between PBPC and BM allografts. The single PBPC leukapheresis product contained 10 times more mononuclear cells, 1.5 times more CD34 + cells, 5.5 times more CD3 + T lymphocytes, 3 times more CD19 + B lymphocytes and 3.8 times more CD14 + monocytes than the single BM harvest. Both CD34 +CD33 + myeloid progenitor cells and CD34 +HLA-DR- long-term reconstituting haemopoietic stem cells were significantly increased in the CD34 + G-CSF-mobilized PBPC compared with the CD34 + BM cells; median 73.1% and 30.4% vs 60.6% and 5.0%, respectively, P < 0.01. The percentage of CD3 + cells coexpressing CD4 (T helper/inducer) was similar in both PBPC and BM allografts, 47.2% and 45.6%, respectively, whereas the percentage of CD3 + cells coexpressing CD8 (T suppressor/cytotoxic) was significantly decreased in PBPC compared with BM; 37.0% vs 55.9%, p < 0.01.

Allogenic transplants with peripheral blood progenitor cells: a report of six cases.

Six patients with high-risk leukaemia received a myeloablative regimen followed by allogeneic peripheral blood progenitor cells transplantation (PBPCT) from an HLA-identical sibling donor. Donors received 10-12 mu g/kg/day of G-CSF subcutaneously for 5 days. G-CSF was well tolerated except for moderate bone pain. Peripheral blood leukapheresis product contained 1-4 times more CD34+ cells and approximately a log more of T lymphocytes than marrow grafts from normal donors. In the two first cases the leukapheresis product was partially depleted of T-lymphocytes using counterflow centrifugation. No growth-factors were administered post-transplant. GVHD prophylaxis consisted of cyclosporin A (CyA) in one case, and CyA and methotrexate in five cases. All patients engrafted with a neutrophil count reaching more than 0.5 x 10(9)/L by day 12 to 21 post-transplant and a platelet count above 20 x 10(9)/L by day 6 to 41 post-transplant. Acute GVHD was clinical grade 0 (n = 2), I (n = 1), II (n = I), grade III (n = I) and grade IV (n = 1). One case presents an extensive chronic cutaneous GVHD and is currently being treated with methylprednisolone. In conclusion, allogeneic transplants using PBPC can be performed safely. This may result in a rapid neutrophil and platelet engraftment, without an apparent increased risk of GVHD.

Failure to Detect Spindle-Shaped Fibroblastoid Cell Progenitors in PBPC Collections

We assessed the development of an adherent stromal layer in peripheral blood progenitor cells (PBPC) collections of 17 patients with solid tumors and hematological malignancies (n = 38), as well as in normal and disease-free bone marrow samples (n = 14). Light-density mononuclear cells from peripheral blood leukapheresis maintained in long term culture (LTC) conditions for at least 3 weeks failed to develop a stromal layer as was observed in all bone marrow samples. We found no differences with regard to CFU-GM growth, CD34+ percentage or cytological morphology. Our results indicate that spindle-shaped fibroblastoid cell progenitors present in the bone marrow are not mobilized towards peripheral blood by chemotherapy and/or growth factor stimulation, contrasting with hematopoietic progenitors, which, as is well known, are able to circulate. The absence of stromal progenitors in PBPC autografts does not appear to be relevant for a successful hematopoietic engraftment.