Cytokine Capture System Research Articles

T cells specific for different latent and lytic viral proteins efficiently control Epstein-Barr virus–transformed B cells

Background aimsEpstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) belong to the most dreaded complications of immunosuppression. The efficacy of EBV-specific T-cell transfer for PTLD has been previously shown, yet the optimal choice of EBV-derived antigens inducing polyclonal CD4+ and CD8+ T cells that cover a wide range of human leukocyte antigen types and efficiently control PTLD remains unclear. MethodsA pool of 125 T-cell epitopes from seven latent and nine lytic EBV-derived proteins (EBVmix) and peptide pools of EBNA1, EBNA3c, LMP2a and BZLF1 were used to determine T-cell frequencies and to isolate T cells through the use of the interferon (IFN)-γ cytokine capture system. We further evaluated the phenotype and functionality of the generated T-cell lines in vitro. ResultsEBVmix induced significantly higher T-cell frequencies and allowed selecting more CD4+IFN-γ+ and CD8+IFN-γ+ cells than single peptide pools. T cells of all specificities expanded similarly in vitro, recognized cognate antigen, and, to a lower extent, EBV-infected cells, exerted moderate cytotoxicity and showed reduced alloreactivity. However, EBVmix-specific cells most efficiently controlled EBV-infected lymphoblastoid cell lines (LCLs). This control was mainly mediated by EBV-specific CD8+ cells with an oligoclonal epitope signature covering both latent and lytic viral proteins. Notably, EBV-specific CD4+ cells unable to control LCLs produced significantly less perforin and granzyme B, probably because of limited LCL epitope presentation. ConclusionsEBVmix induces a broader T-cell response, probably because of its coverage of latent and lytic EBV-derived proteins that may be important to control EBV-transformed B cells and might offer an improvement of T-cell therapies.

Automated isolation of primary antigen‐specific T cells from donor lymphocyte concentrates: results of a feasibility exercise

The safety and clinical efficacy of adoptive transfer of prospectively isolated antigen-specific T cells are well established. Several competing selection methods are available, one of which is based on immunomagnetic enrichment of T cells secreting IFNγ after incubation with the relevant antigen. The proprietary, GMP-conforming selection technology, called 'cytokine capture system' (CCS) is established in many laboratories for the CliniMACS Plus system. It is robust and efficient, but labour-intensive and incompatible with a single-shift working schedule. An automatic immunomagnetic cell processing system, CliniMACS Prodigy ('Prodigy'), including a protocol for fully automatic CCS execution was recently released. Feasibility of clinical-scale CMV-specific T-cell selection using Prodigy was evaluated using leukoapheresis products from five healthy CMV sero-positive volunteers. Clinical reagents and consumables were used throughout. The process required no operator input beyond set-up and QC-sample collection, that is, feasibility was given. An IFNγ-secreting target T-cell population was detectable after stimulation, and >2 log-scale relative depletion of not CMV-reactive T cells in the target population was achieved. Purity, that is the frequency of CMV-reactive T cells among all CD3(+) cells ranged between 64 and 93%. The CCS protocol on Prodigy is unrestrictedly functional. It runs fully automatically beyond set-up and thus markedly reduces labour. The quality of the products generated is similar to products generated with CliniMACS Plus. The automatic system is thus suitable for routine clinical application.

CMV~IMPACT: Results of a Randomized Controlled Trial of Immuno-Prophylactic Adoptive Cellular Therapy following Sibling Donor Allogeneic HSCT

Introduction:Qualitative and quantitative deficiencies in T-cell mediated immunity following allogeneic hematopoietic stem cell transplantation (HSCT) are associated with a high incidence of cytomegalovirus (CMV) infection, which remains an important cause of morbidity and cost, and contributes to mortality. Adoptive cellular therapy (ACT) can potentially expedite reconstitution of CMV-specific immunity and improve outcomes post HSCT. A number of uncontrolled studies have demonstrated proof of concept for such strategies. Previously we presented data from a randomized controlled trial assessing the effect of CMV-specific ACT (Cytovir CMV™) on immune reconstitution following unrelated donor HSCT (CMV~ASPECT). Now we report on a randomized controlled trial to evaluate the incidence of CMV reactivation and safety in a larger cohort of related donor HSCT patients treated with CMV-specific ACT. (CMV~IMPACT: ClinicalTrials.gov NCT 01077908).Methods:The study enrolled CMV-seropositive patients >16 years old, undergoing T-cell depleted (alemtuzumab) HSCT from a matched sibling CMV-seropositive donor, in 14 UK transplant centers. Patients and donors were selected using standard criteria. Patients were randomized into either ACT or control arms. CMV-specific cells were manufactured from a dedicated donor apheresis by direct selection at a central facility using either a StreptamerTM or gamma-capture technique (CliniMACS® Cytokine Capture System, IFN-gamma). Products were manufactured using Streptamers if the donor expressed a suitable HLA allele, or using gamma-capture if not. The 14 patients receiving the latter product are not included in this preliminary analysis. CMV surveillance was performed using quantitative PCR. All groups received standardized antiviral pre-emptive treatment. Cells were cryopreserved at a maximum dose of 5x104CD3+/kg and administered to the patient on day 27/28 post-transplant, regardless of CMV surveillance results (prophylactic administration). Patients with ≥ grade 2 acute GvHD or systemic steroid administration at baseline (D27/28) were withdrawn. Patients were assessed for CMV reactivation based on blinded retrospective assessment by the Chief Investigator. Patients were also assessed for evidence of GvHD and CMV-specific immune reconstitution.Results:As of August 2014, all but one patient had completed the study. There were no clinically apparent differences in serious adverse event (SAE) or acute GvHD events between the two arms. Notably, the number of patients experiencing >1 treatment episode was considerably lower than had been predicted in the control group (26% vs 60% predicted). There were fewer CMV reactivations in the ACT arm compared to the control arm (0.75 vs 1.0/patient), and fewer patients experiencing >1 treatment episode (15% vs 26%), although neither reached statistical significance. There was a trend toward reduced overall treatment duration in the ACT arm (Table 2, p=0.14). Preliminary data are summarized below. A complete and final analysis will be available at the time of the conference.Table1:Patient Disposition/SafetyACTControlEnrolled (withdrawn prior to baseline visit) Safety set Per protocol set (no ACT administered in 10 randomized patients)40 (10) 30 2035 (4) 31 31Reasons for withdrawal · GvHD or steroids at baseline · Other2 82 2No. (%) with new onset acute GvHD (safety set; 100 day follow up)2 (6.7)1 (3.2)No. (%) with at least 1 SAE (safety set)12 (40.0)10 (32.3)Table2:Duration of CMV Treatment (days; per protocol set)ACT (n=20)Control (n=31)pMean (stdv)19.1 (27.8)27.3 (31.3)0.14Median (min:max)11 (0 : 114)25 (0 : 133)Conclusions:In this randomized trial of CMV specific T-cells post-HSCT, adoptive cell therapy (Cytovir CMV™) was not associated with a significant increase in safety events, and preliminary analysis indicates Cytovir CMV administration may be associated with lower rates of CMV reactivation and shorter durations of viral treatment, though the lower than predicted reactivation rate in the control group reduced the power of the trial to demonstrate this at a statistically significant level. DisclosuresCobb:Cell Medica Ltd: Employment. Newton:Cell Medica Ltd: Employment. Thomas:Cell Medica Ltd: Employment. Moss:Cell Medica Ltd: Membership on an entity’s Board of Directors or advisory committees.

Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells.

BackgroundThe adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.MethodsTo obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ+ T cells exceeded 0.03% of CD3+ lymphocytes as determined by IFN-γ cytokine secretion assay.ResultsStarting with low concentration of IFN-γ+ T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ+CD3+ T cells (1.42 × 106, 0.05 × 106, and 1.15 × 106) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8+ than CD4+ memory T cells, both of which were effectively enriched to a total of 81.0% CD8+IFN-γ+ and 38.4% CD4+IFN-γ+ T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.ConclusionsThe generation of antiviral CD4+ and CD8+ T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0336-5) contains supplementary material, which is available to authorized users.

Anti CMV and/or Anti Adenovirus IFN-g-Positive CD4+ CD8+ T Lymphocytes for Treatment of Viral Infections After Allogeneic HSC Transplantation: First Results

AbstractAbstract 1906Reactivation of latent viruses such as cytomegalovirus (CMV) and adenovirus (AdV) is responsible for infections which may be life-threatening in HSCT recipients. In the post-transplantation period, severity and frequency of these infections depend on (a) the degree of donor-recipient HLA incompatibility and (b) the intensity of immunosuppressive therapy used to prevent immunological complications. Antiviral drugs may be partially effective, often toxic and cannot always control those viral infections.T cell immunity plays a major role in the control of viral infections. It has been demonstrated that the transfer of donor T lymphocytes specifically directed against viral antigens is capable of preventing, controlling and clearing viral infection (Feuchtinger T et al., 2004 and 2010). The present project aimed the evaluation of specific, cell-based immunity against CMV and AdV by injection of IFN-g-positive CD4+and CD8+ donor T lymphocytes isolated ex vivo after stimulation with viral peptides. Methods:Our protocol was designed for pediatric or adult patients treated by allogeneic HSCT and matching the following inclusion criteria: (1) biological and/or clinical symptoms of CMV and/or AdV infection 2) no response or contraindication to conventional antiviral treatment and (3) no or low grade pre-existing aGvHD at inclusion (≤ grade II) controlled by corticoids (<1 mg/kg). Antiviral treatments are allowed during the inclusion period.Donor IFN-g-positive T lymphocytes are isolated with the CliniMACS Cytokine Capture System (Miltenyi Biotech) after incubation with viral peptide pools.Primary evaluation criterion is the efficacy of the treatment on CMV viral load 21 days after the first injection. In the event of a negative or partial response and the absence of aGvHD, a second injection may be scheduled.Secondary evaluation criteria are (1) the occurrence of de novo aGvHD or aggravation of existing aGvHD, (2) the evolution of clinical symptoms potentially related to the infection, (3) the demonstration of biological in vivo expansion of injected T lymphocytes (as evidenced by the IFN-g secretion capacity and specific tetramer assays) and (4) for AdV infection, evaluation of efficacy (viral load, in vivo expansion of transfused lymphocytes, clinical symptoms) and the safety (occurrence of aGvHD) of this immunotherapy. Results:From September 2010 to July 2012, 9 patients were included: 3 male adults (46–54 years, 1 CLL, 1 CML and 1 AA, 2 geno- and 1 pheno-identical transplantation) and 6 children (age: 7–25 months, sex ratio F/M: 4/2, 4 FLH, 1 SCID and 1AA, 4 haplo, 1 geno- and 1 pheno-id transplantation).4/9 patients were treated for CMV, 3/9 for AdV and 2/9 for CMV and AdV reactivation. 5/9 patients received 2 cytotoxic T lymphocytes (CTL) injections. Mean number of CD3 IFN-g positive cells injected was 4206/kg (1167–6000/kg) with 55% and 69% of CD4 and CD8 anti CMV-T cells and 56% and 61% of CD4 and CD8 anti AdV T cells respectively.Mean delay of first immunotherapy was 109 days (28–270) after transplantation.2/9 patients were not evaluable due to early death (<21 days post injection) and 1/9 patient died of graft failure 43 days after CTL injection without efficacy on infectious evolution. 6 patients are still alive: 4 with complete, 1 with partial remission of virus replication and 1 recently included, is still under evaluation. An in vivo expansion of transfused CTL was observed (mean expansion was 33 and 35 fold for CD8-IFN-g and CD4-IFN-g positive cells respectively 42 days after injection) in parallel with the decrease of viral load in all alive patients. No aGvHD was detected in the 5/6 evaluated patients. One of 6 presenting cGvH at inclusion need increase of corticotherapy 3 months after second injection of CTL One patient presenting with CMV retinitis received 2 CTL injections without worsening of retina lesions which healed. Conclusion:The CliniMACS Cytokine Capture System allows the isolation of virus-specific T cells in a brief delay (24 hours) with a satisfactory enrichment of both CD4 and CD8 T cells.First results show efficacy of virus-specific T cells injection on viral load without signs of aGvHD in 5/6 evaluable patients. More patients need to be included in this trial in order to confirm these encouraging results. Disclosures:Cambouris:Miltenyi Biotec: Employment.

Effective Treatment of Refractory CMV Reactivation After Allogeneic Stem Cell Transplantation With In Vitro-generated CMV pp65-specific CD8+ T-cell Lines

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8* T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7-14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6-17 × 10(6) cells, comprising 54%-96% CMV pp65-specific CD8 T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8* T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8* T-cell lines could be demonstrated.

New GMP-Grade, Xeno-Component Free Medium for the Activation and Expansion of T Cells

Abstract 4316Therapeutic applications of T cells in immunotherapy have recently gained momentum with the promising results in adoptive transfer of antigen-specific T cells for infectious complications after allogeneic stem cell or solid organ transplantation or for immunotherapy of malignant diseases. Activation and expansion of these cells for clinical application under controlled conditions require GMP-grade reagents including appropriate antibodies, cytokines and media. For standardized, reproducible cell cultivation and ex vivo differentiation procedures, a new serum and xeno-component free, GMP-grade medium for clinical use has been developed: High lot-to-lot consistency has been achieved by eliminating protein components not relevant for T cell expansion leaving human serum albumin as the only protein component.The expansion of T cells in this medium upon polyclonal activation using biotinylated antibodies against CD2, CD3 and CD28 loaded on anti-Biotin coated beads resulted in expansion rates similar to other commercially available serum-free media. Using soluble antibodies against CD3 and CD28, more than 30%-higher expansion rates of viable and functional T cells after 6 days of expansion have been achieved with the new xeno-component free medium compared with other serum-fee media. Transferring the same protocol to a high density cell culture system such as a gas permeable rapid expansion device, high densities of T cells with more than 1.5×107 cells/ mL were reached.The generation of antigen-specific T cells using the Cytokine Capture System (IFN-gamma) and the serum and xeno-component free T cell medium showed similar results regarding purity, recovery and background stimulation compared to the use of a standard basal medium supplemented with 10% human AB serum. For the automation of such complex procedures, a new cell processing device was developed. All steps for the antigen-specific T cell processing, i.e. antigen-specific re-stimulation, magnetic enrichment, and in vitro expansion with this T cell medium are performed in this fully automated device, in a closed system under sterile conditions.In conclusion, the newly developed GMP-grade, serum and xeno-component free T cell medium demonstrated high lot-to-lot consistency and was superior in its performance to other commercially available serum-free media in high density cell culture systems. The new medium can be used to replace human AB serum supplementation for the clinical manufacturing of T cells resulting in easier handling and higher consistency. Disclosures:Assenmacher: Miltenyi Biotec GmbH: Employment. Mockel-Tenbrinck:Miltenyi Biotec GmbH: Employment. Scheffold:Miltenyi Biotec GmbH: Employment. Rauser:Miltenyi Biotec GmbH: Employment. Bohnenkamp:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Odenthal:Miltenyi Biotec GmbH: Employment. Veit:Miltenyi Biotec GmbH: Employment. Kolrep:Miltenyi Biotec GmbH: Employment. Fahrendorff:Miltenyi Biotec GmbH: Employment.

Automated Generation of Antigen-Specific T Cells for Adoptive T Cell Therapy.

AbstractAbstract 1177The adoptive transfer of antigen-specific T cells can be a powerful tool for immunotherapy of malignant diseases or infectious complications after allogeneic stem cell transplantation (Riddell et al., 1992; Heslop et al. 2010). Human adenovirus (AdV), Epstein-Barr virus (EBV) or cytomegalovirus (HCMV) infections are frequent and often life-threatening complications post allogeneic stem cell transplantation. To reduce the time required to isolate antigen-specific T cells for adoptive transfer we have developed a method to isolate IFN-gamma-secreting CD4+ as well as CD8+ T cells after antigen-specific restimulation, the Cytokine Capture System IFN-gamma (CCS). Several preclinical studies demonstrate the efficient enrichment of functional CD4+ and CD8+ T cells specific for HCMV (Rauser et al., 2004), EBV (Hammer et al., 2007) or AdV (Feuchtinger et al. 2008) using the CCS. First clinical data of adoptively transferred HCMV-, EBV- or AdV-specific T cells into patients post allogeneic stem cell transplantation are very encouraging (Feuchtinger et al., 2010; Moosmann et al., 2010; Feuchtinger et al., 2006) with low T cell doses infused varying from 1–97×10e3 cells/kg. We have now developed a cell processing device for the automation of the CCS procedure. Antigen-specific stimulation, labeling with the CCS reagents, washing steps, cytokine capture, magnetic enrichment and potentially expansion of the isolated cells are performed fully automated in a closed system. At the beginning of the procedure all components including the cellular starting product, antigen(s), reagents, buffer, and media are connected to a sterile single-use closed system processing set in the device. Due to usage of sterile filters and sterile docking the whole process runs under sterile conditions. The cellular end product can be obtained in the medium/buffer of choice, with the desired cell concentration and volume. The cellular starting product can be leukapheresis or bone marrow and the yield of antigen-specific T cells depends on the frequency of IFN-gamma producing cells. When starting with 1×10e9 cells 1–20×10e5 HCMV-specific T cells could be isolated. Cell processing is possible overnight and the isolated cells might be used directly after enrichment or after a phase of in vitro expansion. Using this cell processing device, IFN-gamma secreting HCMV-specific T cells were enriched to the same purity (>80% IFN-gamma secreting CD4+ and CD8+ T cells) as with the semi-automated procedure. Cell loss during the procedure is markedly reduced, leading to an increased yield of IFN-gamma positive cells. An improved viability was observed resulting in better expansion rates. In conclusion, the automation in a closed system enables the fast and robust generation of antigen-specific T cells for adoptive therapy and will reduce clean room requirements. Disclosures:Fahrendorff:Miltenyi Biotec GmbH: Employment. von Oppen:Miltenyi Biotec GmbH: Employment. Rauser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Biehl:Miltenyi Biotec GmbH: Employment. Miltenyi:Miltenyi Biotec GmbH: Membership on an entity's Board of Directors or advisory committees.