ALBERT

Description: Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4+ and CD8+ CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-γ (IFN-γ) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 × 108 combined CD4+ and CD8+ CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-γ production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4+ and CD8+ T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4+ and CD8+ T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4+ and CD8+ CMV-specific T cells under conditions mimicking good manufacturing practice. (Blood. 2004; 103:3565-3572)

Description: Abstract 4316 Therapeutic 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.

Description: Abstract 1177 The 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.

Description: Reconstitution of human cytomegalovirus (HCMV)–specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-γ release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8+ T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide–specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen–specific CD4+ T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35-〉180 days). Peak counts of peptide-specific CD8+ T cells ranged from 0.14 to 60.6 cells/μL; those of protein-specific CD4+ T cells ranged from 0.64 to 18.97 cells/μL. Reconstitution of HCMV-peptide–specific CD8+ T cells and protein-specific CD4+ T cells was associated with clearance of HCMV infection (r2 = 0.89, P 

Description: Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.

Description: Introduction CD19 CAR-T cell products were recently approved as therapy for B-lineage malignancies. We initiated an IIT trial where manufacture of CAR-T cells was performed locally using a unique CD19 CAR with potent anti-leukemic effects. Patients and methods A total of 37 pts with relapsed/refractory B-acute lymphoblastic leukemia (12 female, 25 male, median age 10 y) were screened, 27 pts were enrolled for a trial, 10 were eligible for compassionate use of CD19 CAR-T cell therapy. Sixteen patients had relapsed B-ALL after haploidentical HSCT, 19 pts refractory relapse, 2 induction failure, 13 patients had previous blinatumomab infusion. Eighteen patients had 〉20% blast cells, median bone marrow leukemia burden for patients with full blown disease was 89%, 19 pts had minimal residual disease (MRD) 〉0.1% in BM, 3 had skeletal involvement with multiple mass lesions, one had CNS involvement. The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells. The automated production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct and transduced with lentiviral vector expressing the CD19CAR gene (second generation CD19.4-1BB zeta with alternate transmembrane domain derived from the TNF superfamily) (Lentigen, Miltenyi Biotec company). T cells were expansion over 10 days in the presence of serum-free TexMACS GMP Medium supplemented with MACS GMP IL-7 and IL-15. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab at 8mg/kg before CAR-T cell infusion. Patients did not receive HSCT as consolidation after CAR-T therapy. Results Thirty-five manufacturing cycles were successful. Median transduction efficacy was 60% (20-80). Median expansion of T cells was x 46 (18-51). CD4:CD8 ratio in the final product was 0.73. The cell products were administered at a dose of 3*106/kg of CAR-T cells in 4 pts, 1*106/kg in 9 pts, 0.5*106/kg in 14 pts, 0.1*106/kg in 8 pts. Two patients received 0.1*106/kg of CAR-T cells produced from haploidentical donors. The cytokine release syndrome (CRS) occurred in 22 (59%) pts and was mostly mild and moderate: grade I - 15 pts, grade II- 4 pts, grade III - 2 pt, grade IV - 1 pt. CAR-T cell related encephalopathy occurred in 15 (40%). Grade I-II neurotoxicity developed in 10 pts, grade III - in 2 pt, grade IV - 1 pt, grade V - 2 pt. In one patient with grade V neurotoxicity concomitant K. pneumonia encephalitis was documented. Severe (grade 3-5) CRS and neurotoxicity were associated exclusively with large leukemia burden (〉20% in the bone marrow) at enrollment, p=0,002. Thirty-one patient was evaluable for response at day 28. Four pts had persistent leukemia. In 27 (87%) cases Flow MRD-negative remission was achieved. Disease relapse after initial response was registered in 9 (33%) cases (7 patients had CD19 negative, 2 had CD19 positive relapse). At the moment of reporting, 10 patients have died (3 due to sepsis, 1 due to brain edema, 1 due to brain edema and K. pneumonia encephalitis, 5 due to progression of disease or relapse). Twenty-seven pts are alive, 19 in complete remission with a median follow up of 223 days (41-516 days). Conclusion CliniMACS Prodigy TCT process is a robust CAR-T cell manufacturing platform that enables rapid and flexible provision of CAR-T cells to patients in need. Significant toxicity of CD19 CAR-T cells was associated exclusively with high leukemia burden at enrollment. In the absence of HSCT consolidation relapse rate exceeds 30%. Disclosures Schneider: Lentigen Technology, A Miltenyi Biotec Company: Employment. Preussner:Miltenyi Biotec: Employment. Rauser:Miltenyi Biotec: Employment. Orentas:Lentigen Technology Inc., a Miltenyi Biotec Company: . Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Maschan:Miltenyi Biotec: Other: lecture fee.