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Mutated isocitrate dehydrogenase 1 (IDH1) defines a molecularly distinct subtype of diffuse glioma1–3. The most common IDH1 mutation in gliomas affects codon 132 and encodes IDH1(R132H), which harbours a shared clonal neoepitope that is presented on major histocompatibility complex (MHC) class II4,5. An IDH1(R132H)-specific peptide vaccine (IDH1-vac) induces specific therapeutic T helper cell responses that are effective against IDH1(R132H)+ tumours in syngeneic MHC-humanized mice4,6–8. Here we describe a multicentre, single-arm, open-label, first-in-humans phase I trial that we carried out in 33 patients with newly diagnosed World Health Organization grade 3 and 4 IDH1(R132H)+ astrocytomas (Neurooncology Working Group of the German Cancer Society trial 16 (NOA16), ClinicalTrials.gov identifier NCT02454634). The trial met its primary safety endpoint, with vaccine-related adverse events restricted to grade 1. Vaccine-induced immune responses were observed in 93.3% of patients across multiple MHC alleles. Three-year progression-free and death-free rates were 0.63 and 0.84, respectively. Patients with immune responses showed a two-year progression-free rate of 0.82. Two patients without an immune response showed tumour progression within two years of first diagnosis. A mutation-specificity score that incorporates the duration and level of vaccine-induced IDH1(R132H)-specific T cell responses was associated with intratumoral presentation of the IDH1(R132H) neoantigen in pre-treatment tumour tissue. There was a high frequency of pseudoprogression, which indicates intratumoral inflammatory reactions. Pseudoprogression was associated with increased vaccine-induced peripheral T cell responses. Combined single-cell RNA and T cell receptor sequencing showed that tumour-infiltrating CD40LG+ and CXCL13+ T helper cell clusters in a patient with pseudoprogression were dominated by a single IDH1(R132H)-reactive T cell receptor., A phase 1 clinical trial provides evidence that a vaccine against mutant IDH1 is safe and produces a T helper immune response in patients with glioma.

High-dose chemotherapy (HD-CHT) and autologous blood stem cell transplantation (ABSCT) represent the standard of care in multiple myeloma (MM) for transplantation-eligible patients. Up to 3 HD-CHT/ABSCT treatments may be administered during the course of disease, including during late-onset relapse. Transplantation centers routinely collect more than 1 peripheral blood stem cell (PBSC) graft; however, subsequent HD-CHT/ABSCT treatments are often not performed, for various reasons. Currently, little is known about the actual utilization rate of stored PBSCs. The collection, storage, and disposal of PBSC products was analyzed in a large cohort of patients with MM (n = 1114) over a 12-year period with a minimum follow-up of 6 years. The final dataset analysis was performed in March 2019, which was set as the reference date. Based on institution-specific charges, the costs for PBSC collection, processing, and storage were estimated. The median number of sufficient PBSC transplantations per patient was 3 (range, 0 to 6), which were stored in a median of 3 (range, 1 to 11) cryopreserved bags (overall, n = 3644). A total of 95% of all patients (n = 1059) underwent at least 1 HD-CHT/ABSCT treatment. However, multiple ABSCTs were performed in 51% of the patients (n2/3 ABSCTs = 538), and only 14% of the patients underwent ABSCT 3 times (n3 ABSCTs = 149). Only a small proportion of collected PBSC bags (5%; n = 109) were used after being stored for longer than 5 years. Overall, 23% of the products (n = 830) were discarded, and 16% (n = 566) were kept in storage until the reference date. From a retrospective standpoint, the collected and discarded (definitively not used) or stored (potentially not used) cryostored PBSCs were associated with considerable costs for long-term cryostorage of approximately €1,600,000. We identified considerable discrepancies between the collection/storage and utilization of PBSCs. This is associated with significant efforts and costs on the one hand; on the other hand, disposal may raise legal and ethical questions. Therefore, we implemented comprehensive guidelines for the systematic reevaluation of stored PBSC grafts at our institution.

Although CD19-directed chimeric antigen receptor (CAR) T cells have been successfully used after a preceding allogeneic stem cell transplant (alloHCT) in patients with acute lymphoblastic leukemia, little is known about the feasibility and outcome of CAR T cell treatment in patients who have been previously allotransplanted for lymphoma. In a single-center retrospective analysis, course and outcome of all allografted patients treated with CD19 CAR constructs for B cell lymphoma between October 2018 and November 2019 were studied. CAR therapy consisted either of a third-generation CAR (HD-CAR-1) or of commercially manufactured axicabtagene ciloleucel (axi-cel; Gilead, Santa Monica, U.S.). Altogether, 10 CAR T cell dosings using recipient leukapheresis products were performed in 8 patients: 4 patients (2 mantle cell lymphoma, 2 chronic lymphocytic leukemia) received 6 dosings with HD-CAR-1 and 4 patients (all with diffuse large B cell lymphoma) received 4 dosings with axi-cel. Overall, 6 of 8 patients (75%) responded. CAR treatment was well tolerated with grade ≥ 3 cytokine release syndrome and neurotoxicity each being observed after 1 of 10 dosings. A single patient had moderate chronic graft-versus-host disease. Of note, 3 of 4 patients who received axi-cel had ongoing grade ≥ 3 cytopenia 3 months postdosing, whereas prolonged cytopenia was not observed in 9 alloHCT-naive patients who received axi-cel during the same time period. In conclusion, CAR T cell treatment from recipient-derived leukapheresis products after a prior alloHCT appears to be feasible, effective, and safe in patients with B cell lymphoma. Protracted cytopenia after axi-cel treatment is a matter of concern and requires further exploration.

Background Beti-cel ex vivo gene therapy integrates a modified HBB gene into hematopoietic stem cells of patients with TDT, aiming to enable lifelong, stable production of functional adult hemoglobin (Hb). The efficacy and safety of the treatment have been demonstrated in a total of 63 patients treated across 4 clinical trials (HGB-204,-HGB-205, HGB-207, and HGB-212). Here, we present the first patient who received beti-cel outside of the clinical trial setting, a 14-year-old male with a β 0/β + (IVS-1-6) genotype. Methods Following hematopoietic stem cell collection via granulocyte-colony stimulating factor plus plerixafor mobilization and apheresis, CD34+ cells were transduced with the BB305 lentiviral vector encoding HbA T87Q. The patient received hypertransfusion before mobilization and conditioning, maintaining a pre-transfusion Hb level of >11 g/dL. Six days prior to beti-cel infusion, single-agent busulfan myeloablation was initiated (16 single doses at 0.8 mg/kg body weight; 3.2 mg/kg/24 h) with concomitant clonazepam (see Table for treatment timeline). Ursodeoxycholic acid therapy was continued as hepatic veno-occlusive disease (VOD) prophylaxis through inpatient treatment. Results The patient was diagnosed with TDT at the age of 2 years in his home country and has been treated in Germany since the age of 9. Regular transfusion therapy was initiated soon after diagnosis (Table). Aged 9, the patient was started on desferasirox for iron elimination therapy. His annualized red blood cell (RBC) transfusion volume was 174 ml/kg in 2018 and 185 ml/kg in 2019, maintaining his pre-transfusion Hb at or above 9 g/dl. No HLA-related donor was available for allogeneic transplant. At informed consent, the patient was 13 years old and met the eligibility criteria for beti-cel treatment as outlined in the summary of product characteristics (SmPC). The patient was physically fit, with a 90% Lansky score and regular participation in school sports, but reported physical limitations when running extensively. The patient underwent a thorough assessment before admission (Table), which did not reveal any remarkable abnormalities except TDT-related splenomegaly and signs of slight iron overload (liver iron content, 2.0 mg/g dry weight [normal range, 0.17-1.8]). On 11/Feb/2021, the patient was infused with 5.1 × 10 6 CD34+ cells/kg. The patient received 4 RBC and 8 platelet transfusions following infusion until Day 13 and 27, respectively (Table). Neutrophil and platelet engraftment occurred on day 27 post beti-cel infusion. The patient was discharged from inpatient treatment the same day, in excellent general condition, with 90% Lansky score, Hb of 8.2 g/dl, a reticulocyte count of 9.3%, a total white cell count of 1.55/nl, a neutrophil count of 0.75/nl, and a platelet count of 24/nl. At last follow-up (+100 days), the patient felt well and exhibited normal exercise tolerance. He has received neither red blood cell nor platelet transfusions or chelation therapy since discharge. Total Hb was 11.8 g/dl (Table). Granulocytes and lymphocytes had recovered to normal levels. The patient showed continued, albeit slowly improving, thrombocytopenia (platelet count, 31/nl [29/nl at +60 days]), consistent with previous observations after beti-cel therapy. Myeloablation and beti-cel infusion were tolerated well. Adverse events post infusion were febrile neutropenia, elevated C-reactive protein levels, pruritus, gingivitis, mild mucositis, and vertigo, consistent with the SmPC. At +23 and +26 days, the patient experienced transient subjective hearing loss (quickly resolved). No VOD events occurred. Conclusions This is the first real-world patient with TDT treated with beti-cel therapy. The treatment regimen had a tolerability profile consistent with that of mobilization, apheresis, and busulfan myeloablation, matching clinical trial observations. Following treatment, this 14-year-old patient reached a total Hb of 11.8 g/dL at +100 days without requirement of red cell transfusions and continues to exhibit prolonged but slowly improving and asymptomatic thrombocytopenia. Figure 1 Figure 1. Disclosures Schmitt: TolerogenixX Ltd: Current Employment; Therakos/Mallinckrodt: Research Funding; Hexal: Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant. Schmitt: Bluebird Bio: Other: Travel grants; Novartis: Other: Travel grants, Research Funding; TolerogenixX: Current holder of individual stocks in a privately-held company; Apogenix: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants. Kulozik: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria; BioMedX: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

In multiple myeloma, local radiation therapy (RT) of osseous lesions before peripheral blood stem cell (PBSC) mobilization is assumed to impair the PBSC mobilization and collection. However, the results of previously published studies are inconsistent and do not evaluate detailed metrics of RT and PBSC outcome parameters. In total, 352 patients undergoing PBSC mobilizations and RT in first-line treatment were evaluated. Patients were grouped into RT (n = 283) and no RT (n = 69) before PBSC mobilization. Except for the International Staging System score, both groups were homogeneous regarding the first diagnosis characteristics, first-line treatments, and response parameters. RT metrics (RT yes versus no, volume of irradiated hematopoietic bone marrow [BM], biologically equivalent doses in 2 Gy fractions [EQD2]) were analyzed for the following PBSC outcome parameters: achievement of the PBSC collection goal, CD34+ cell collection yield, duration of the mobilization phase, and number of leukapheresis (LP) sessions to reach the collection goal. No statistically significant differences in the percentage of collection failures to reach at least 3 sufficient PBSC transplants were identified comparing patients with (n = 32 [11%]) and without RT (n = 4 [6%]) before PBSC mobilization (P = .265). However, patients with RT before PBSC mobilization showed a significant prolongation of the PBSC mobilization (median 1 day, P =.026) and required a higher number of LP sessions to reach the collection goal (median 1 LP, P.001) compared with patients who received RT after PBSC mobilization. Moreover, patients with RT before PBSC mobilization reached a significantly lower CD34+ cell collection result (mean 8.94 versus 9.81 × 10

Simple Summary Chimeric antigen receptor T (CAR-T) cells have become clinical practice for the treatment of acute lymphoblastic leukemia and non-Hodgkin lymphoma. The aim of this retrospective study was to assess infection complications after lymphodepletion and CAR-T cell therapy. Infections were commonly detected, but manageable in most cases. Fast and appropriate identification as well as treatment were critical, especially in this very vulnerable patient group. Effective strategies to prevent infections as well as adequate medical management also include standardized prophylaxis and additional supportive therapy. Abstract Chimeric antigen receptor T (CAR-T) cell therapy has proven to be very effective in patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). However, infections—related either due to lymphodepletion or the CAR-T cell therapy itself—can result in severe and potentially life-threatening complications, while side effects such as cytokine release syndrome (CRS) might complicate differential diagnosis. Sixty-seven dosings of CAR-T cells in sixty adult patients with NHL (85%) and ALL (15%) receiving CAR-T cell therapy were assessed for infectious complications. Almost two-thirds of patients (61%) developed fever following lymphodepletion and CAR-T cell dosing. Microbiological or radiological findings were observed in 25% of all cases (bacterial 12%, viral 5%, fungal 8%). Inpatient infections were associated with more lines of therapy and more severe CRS. However, overall serious complications were rare after CAR-T therapy, with one patient dying of infection. Pathogen detection after inpatient stay was infrequent and mostly occurred in the first 90 days after dosing. Infections in CAR-T cell treated patents are common. Fast and suitable identification and treatment are crucial in these heavily pretreated and immunocompromised patients. In most cases infectious complications are manageable. Nonetheless, standardized anti-infective prophylaxis and supportive therapy are mandatory to reduce morbidity and mortality in CAR-T cell therapy.

CD19-directed chimeric antigen receptor (CAR) T-cell treatment has evolved as standard of care (SOC) for multiply relapsed/refractory (R/R) large B-cell lymphoma (LBCL). However, its potential benefit over allogeneic hematopoietic cell transplantation (alloHCT) remains unclear. We compared outcomes with both types of cellular immunotherapy (CI) by intention to treat (ITT). Eligble were all patients with R/R LBCL and institutional tumor board decision recommending SOC CAR T-cell treatment between July 2018 and February 2020, or alloHCT between January 2004 and February 2020. Primary end point was overall survival (OS) from indication. Altogether, 41 and 60 patients for whom CAR T cells and alloHCT were intended, respectively, were included. In both cohorts, virtually all patients had active disease at indication. CI was recommended as part of second-line therapy for 21 alloHCT patients but no CAR T-cell patients. Median OS from indication was 475 days with CAR T cells vs 285 days with alloHCT (P = .88) and 222 days for 39 patients for whom alloHCT beyond second line was recommended (P = .08). Of CAR T-cell and alloHCT patients, 73% and 65%, respectively, proceeded to CI. After CI, 12-month estimates for nonrelapse mortality, relapse incidence, progression-free survival, and OS for CAR T cells vs alloHCT were 3% vs 21% (P = .04), 59% vs 44% (P = .12), 39% vs 33% (P = .97), and 68% vs 54% (P = .32), respectively. In conclusion, CAR T-cell outcomes were not inferior to alloHCT outcomes, whether measured by ITT or from CI administration, supporting strategies preferring CAR T cells over alloHCT as first CI for multiply R/R LBCL.

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted.

Background: T lymphocyte collection through leukapheresis is an essential step for chimeric antigen receptor T (CAR-T) cell therapy. Timing of apheresis is challenging in heavily pretreated patients who suffer from rapid progressive disease and receive T cell impairing medication. Methods: A total of 75 unstimulated leukaphereses were analyzed including 45 aphereses in patients and 30 in healthy donors. Thereof, 41 adult patients with Non-Hodgkin&rsquo, s lymphoma (85%) or acute lymphoblastic leukemia (15%) underwent leukapheresis for CAR-T cell production. Results: Sufficient lymphocytes were harvested from all patients even from those with low peripheral lymphocyte counts of 0.18/nL. Only four patients required a second leukapheresis session. Leukapheresis products contained a median of 98 &times, 108 (9 - 341 &times, 108) total nucleated cells (TNC) with 38 &times, 108 (4 - 232 &times, 108) CD3+ T cells. Leukapheresis products from healthy donors as well as from patients in complete remission were characterized by high TNC and CD3+ T lymphocyte counts. CAR-T cell products could be manufactured for all but one patient. Conclusions: Sufficient yield of lymphocytes for CAR-T cell production is feasible also for patients with low peripheral blood counts. Up to 12&ndash, 15 L blood volume should be processed in patients with absolute lymphocyte counts &le, 1.0/nL.

BACKGROUND: Preclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor. METHODS: In this phase I trial, patients received either 1.5 × 10(6) MICs per kg BW on day –2 (n = 3, group A), or 1.5 × 10(8) MICs per kg BW on day –2 (n = 3, group B) or day –7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360. RESULTS: MIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 10(10) donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19(+)CD24(hi)CD38(hi) transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature. CONCLUSION: MIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs. TRIAL REGISTRATION: EudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220. FUNDING: Federal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany.

Background High-dose (HD) chemotherapy followed by autologous blood stem-cell transplantation (ASCT) is the standard treatment for multiple myeloma (MM) patients. However, the collection of sufficient peripheral blood stem cell (PBSC) grafts can be challenging, and the question arises whether reinfusion of low-dose grafts will lead to a hematopoietic recovery. Methods The hematopoietic recovery of 148 MM patients who underwent HD melphalan chemotherapy and received PBSC transplants with varying CD34+ cells doses (3–4 × 106 [n = 86], 2–2.5 × 106 [n = 53], 6 [n = 9] per kg body weight [bw]) was analyzed in this retrospective single-center study. Results All patients reached hematopoietic reconstitution, even those who received 6 CD34+ cells/kg bw. 62 (42%) patients received granulocyte-colony-stimulating factor (G-CSF). The median duration to leukocyte recovery ≥1.0 × 109/L was 12 days in every group. The median duration to platelet recovery ≥20 × 109/L was 11, 13 and 13 days, respectively. In the multivariate analysis, a low number of reinfused CD34+ cells was associated with prolonged time until leukocyte reconstitution (p = 0.010, HR 0.607) and platelet recovery (p Conclusion In conclusion, reinfusion of low- and even very-low-dose PBSC grafts leads to sufficient hematopoietic reconstitution. No severe adverse events were observed during or after HD chemotherapy and ASCT in the analyzed cohort. While the impact of CD34+ cell dose is marginal, G-CSF significantly accelerates the leukocyte recovery.

In patients with steroid-refractory/resistant graft-versus-host disease (GvHD), extracorporeal photopheresis (ECP) has been identified as a promising therapeutic strategy due to its safety profile and convincing clinical response rates. While previous studies have reported on the role of Th1, Th2 and Treg cells in ECP therapy, further comprehensive analysis of T helper cells is necessary to provide better understanding of the underlying mechanisms. In this study, we thus investigated the influence of long-term ECP treatment on both Th cells as well as on immune checkpoint molecules and apoptosis. Overall, we investigated 27 patients with GvHD treated by ECP therapy, containing 13 patients with acute GvHD and 14 patients with chronic GvHD. 10/13 (76.9%) of aGvHD and 9/14 (64.3%) of cGvHD patients clinically responded to ECP therapy. Three (23.1%) patients reached CR and seven (53.8%) patients achieved PR under ECP treatment in patients with aGvHD, while in patients with cGvHD CR was achieved in one (7.1%) and PR in eight (57.1%) patients. Stabilization of disease was observed in five (35.7%) patients. On an immunological level, aGvHD, cGvHD and healthy donors (HD) patients showed different profiles of Th populations. In GvHD patients, significantly higher levels for Th2 (aGvHD vs. HD: 36.26% vs. 13.88%, p = 0.014; cGvHD vs. HD: 30.71% vs. 13.88%, p = 0.026), Th17 (aGvHD vs. HD: 19.21% vs. 7.49%, p = 0.038), Th22 (aGvHD vs. HD: 1.22% vs. 0.11%，p = 0.011; cGvHD vs. HD: 0.64% vs. 0.11%, p = 0.012) and GM-CSF + Th cells (aGvHD vs. HD: 1.15% vs. 0.14%, p = 0.022; cGvHD vs. HD: 0.84% vs. 0.14%，p = 0.012) and clearly lower levels for T follicular helper (Tfh) cells including Th1- (aGvHD vs. HD: 0.37% vs. 2.04%, p = 0.002; cGvHD vs. HD: 1.28% vs. 2.04%, p = 0.03) and Th2-like cells (aGvHD vs. HD: 1.17% vs. 4.19%, p = 0.033) were observed in comparison to HDs. This suggests Th cells account for a crucial role in GvHD pathogenesis. ECP therapy was able to accelerate recovery of Tfh cells, including Th1- (Time point 1 vs. Time point 2 in aGvHD: 0.37% vs. 0.97%, p = 0.028), Th2- (T1 vs. T2 in aGvHD: 1.17% vs. 1.35%, p = 0.049; T1 vs. T2 in cGvHD: 2.08% vs. 3.15%, p = 0.048) and Th17-like Tfh cells (T1 vs. T2 in aGvHD: 1.82% vs. 2.70%, p = 0.034), facilitating immune reconstitution after allo-HSCT and thereby alleviating GvHD. Clinical response in aGvHD patients was strongly associated with elevation of Th22 cells by ECP therapy showing 51% upregulation (T1 vs. T2: 0.86% vs. 1.3%，p=0.007). In addition, we also found that the expression of the Fas receptor on effector T cells could be further upregulated with ECP treatment (T1 vs. T2 in cGvHD: 49.65% vs. 56.46%, p = 0.011), which increases the susceptibility of these effectors T cells to Fas-mediated apoptosis. This could lead to the elimination of these overactivated effector T cells and the perseverance of immunological tolerance by triggering activation-induced cell death. In addition, a reduction of Tim-3-expressing effector T cells, which are associated with the severity of GvHD, with ECP therapy (T1 vs. T2 for aGvHD: 18.09% vs. 15.10%, p = 0.044) was discovered. In conclusion, ECP therapy exerts immunomodulatory effects by promoting balanced immune reconstitution and inducing immune tolerance, making it an attractive and promising treatment option for patients with GvHD. Disclosures Schubert: Gilead: Consultancy. Hegenbart: Alnylam: Honoraria; Akcea: Honoraria; Janssen: Consultancy, Research Funding; Prothena: Research Funding; Pfizer: Consultancy, Honoraria. Schönland: Prothena: Honoraria, Other: Travel grants; Takeda: Honoraria, Other: Travel grants; Pfizer: Honoraria; Janssen: Honoraria, Other: Travel grants, Research Funding; Sanofi: Research Funding. Müller-Tidow: Pfizer: Research Funding; Bioline: Research Funding; Janssen: Consultancy, Research Funding. Dreger: Riemser: Consultancy, Research Funding, Speakers Bureau; Bluebird Bio: Consultancy; Roche: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau. Schmitt: MSD: Membership on an entity's Board of Directors or advisory committees; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants; TolerogenixX: Current holder of individual stocks in a privately-held company; Bluebird Bio: Other: Travel grants; Novartis: Other: Travel grants, Research Funding; Apogenix: Research Funding. Schmitt: Therakos/Mallinckrodt: Research Funding; TolerogenixX Ltd: Current Employment; Jazz Pharmaceuticals: Other: Travel grant; Hexal: Other: Travel grant.

BACKGROUND Granulocyte–colony-stimulating factor (G-CSF) originators such as filgrastim (Neupogen) and lenograstim (Granocyte) are widely used for peripheral blood stem cell (PBSC) mobilization. In recent years, biosimilar agents have been approved for the same indications. The aim of this retrospective study was to compare the mobilization efficiency of the three G-CSF variants originator filgrastim, lenograstim, and the biosimilar Filgrastim Hexal in a homogeneous group of multiple myeloma (MM) patients in first-line therapy. STUDY DESIGN AND METHODS Overall mobilization data of 250 patients with MM were included. Of these patients, 74 (30%), 131 (52%), and 45 (18%) were mobilized with originator filgrastim, biosimilar Filgrastim Hexal, or lenograstim, respectively, at a dose of 5 to 10 µg/kg body weight subcutaneously starting from Day 5 after chemomobilization with CAD (cyclophosphamide, doxorubicin, dexamethasone) until completion of PBSC collection. RESULTS All but one patient reached the collection goal of a minimum of at least 2 × 106 CD34+ cells/kg body weight during a median of one (range, one to three) leukapheresis session. No significant differences in CD34+ mobilization and collection yields between the filgrastim-mobilized (median, 10.5; range, 2.7-40.4), Filgrastim Hexal–mobilized (median, 9.9; range, 0.2-26.0), and lenograstim-mobilized (median, 10.7; range, 3.1-27.9 CD34+ cells × 106/kg body weight) patients were observed. CONCLUSION Concerning the clinically relevant efficiencies of PBSC mobilization and in terms of reaching the individual collection target, this retrospective study did not detect any significant differences between the three G-CSF variants in the analyzed patient cohort.

BACKGROUND: In a previous study we have shown that CD19-directed chimeric antigen receptor (CAR)-T cells do not appear to be inferior to alloHCT when used as standard cellular immunotherapy (CI) for patients with multiply r/r LBCL (EBMT 2020). The purpose of the present follow-up analysis was to further compare the risk profile of the 2 cohorts by applying the EASIX score (lactate dehydrogenase (U/L) × creatinine (mg/dL)/thrombocytes (109 cells per L)), and to assess if EASIX could be used as outcome predictor in patients with r/r LBCL undergoing CAR-T and alloHCT, respectively. METHODS: Eligible were all patients referred to our institution with relapsed/refractory (R/R) DLBCL and a tumor board decision recommending treatment with CAR-T cells between 07/2018 and 02/2020 and those recommending allogeneic donor search between 2004 and 2019. Patients with DLBCL transformed from CLL were excluded. EASIX was evaluated retrospectively using uni- and multivariable analyses (with regards to age, gender and number of failed therapy lines) and mortality using Cox regression analyses. RESULTS: 41 patients intended for CAR-T cells and 60 patients intended for alloHCT were included. In both cohorts nearly all patients had active disease at indication. Cohorts were comparable for sex, time from diagnosis, ZUMA1 eligibility, and PS, but CAR-T patients tended to be older (median 56 vs 51 years, p=0.093), and had more often primary refractory and bulky disease (p=0.004 and p=0.04, respectively). Median EASIX score across both cohorts was 1.50 (0.27-70.5), with significantly higher scores in the CART group both at indication (EASIX-ind; median 1.79 and 1.22 for CAR-T and alloHCT, respectively, p=0.031) and at conditioning for CI (EASIX-pre, median 2.24 vs 1.26, p=0.005). Median OS from indication was 475d for the CAR-T cohort vs 285d for the alloHCT cohort (p=0.88). On multivariate analysis, EASIX-ind was significantly associated with adverse OS if alloHCT was intended (HR per 2fold increase 1.43, 95%CI 1.08-1.90, p=0.013), but not if CAR-T was intended (HR per 2fold increase 1.16, 95%CI 0.88-1.53, p=0.3). After CI, 12-month estimates for NRM, relapse incidence, PFS, and OS for CAR-T vs alloHCT were 3% vs 21% (p=0.04), 59% vs 44% (p=0.12), 39% vs 33% (p=0.97), and 68% vs 54% (p=0.32). EASIX-pre predicted overall survival (OS) in both CAR-T (HR per 2fold increase 2.11, 95%CI 1.21-3.7, p=0.009) and alloHCT (HR per 2fold increase 3.69, 95%CI 1.54-8.31, p=0.003) cohorts. In the alloHCT group, the EASIX effect was largely driven by higher NRM risk with increasing EASIX-pre, while in the CAR-T group poorer OS with increasing EASIX-pre was largely relapse-related. CONCLUSIONS: In patients undergoing CI for r/r LBCL, EASIX measured prior to conditioning can predict mortality after both CAR-T and alloHCT. If applied already at indication for CI, the predictive capacity of EASIX is weaker and no longer significant if CAR-T is intended. Further studies for validation of this data appear to be warrantable. Disclosures Schmitt: MSD: Membership on an entity's Board of Directors or advisory committees, Other: PI of clinical trials on letermovir; TolerogenixX Ltd: Other: Co-Founder and shareholder; Hexal: Other: Travel grants , Research Funding; Apogenix: Research Funding; Kite: Other: Travel grants, educational activities and conferences; Novartis: Other: educational activities and conferences, Research Funding. Dietrich:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Schmitt:Hexal: Other: Travel grants ; TolerogenixX LtD: Other: Co-founder, Part-time employee ; Therakos/Mallinckrodt: Research Funding; Jazz Pharmaceuticals: Other: Travel grants . Dreger:Neovii: Research Funding; Roche: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; Gilead: Consultancy, Speakers Bureau; AstraZeneca: Consultancy; AbbVie: Consultancy, Speakers Bureau.

Introduction: Recently, we identified a huge discrepancy between the collection practice and the actual utilization of cryopreserved peripheral blood stem cells (PBSCs) for high-dose chemotherapy (HDCT) and autologous blood stem cell transplantation (ABSCT). Specifically, patients with Burkitt lymphoma, acute leukemia, and myeloproliferative neoplasms (MPN) were frequently not referred for ABSCT after successful PBSC collection. Objective: The aim of this study was to identify variables that are associated with the non-utilization of PBSC grafts. Methods: We retrospectively analyzed the collection, storage, and disposal of PBSC grafts in Burkitt lymphoma (n = 18), acute lymphoblastic leukemia (ALL, n = 22), MPN (n = 18), and acute myeloid leukemia (AML, n = 71) patients. Patients who underwent autologous PBSC collection at 2 collection and transplantation centers between 2001 and 2012 were included and followed up until 2016. Results: None of the Burkitt lymphoma patients were referred for ABSCT. Only in 1 (6%) patient, the graft was discarded after the patient’s death. In all other patients (n = 17, 94%), the grafts were stored independently of the patient’s status (death, n = 4, 22%; no follow-up, n = 6, 33%; no indication for ABSCT given, n = 7, 39%). In ALL patients, 4 (18%) patients underwent ABSCT after a median follow-up of 74 (1–182) months. In the remaining patients, PBSC grafts were either discarded (8 patients, 36%) or stored until the reference date (10 patients, 45%). Seven of 18 MPN patients (39%) underwent ABSCT. ABSCT was performed in 24 (34%) AML patients. In 20 (28%) patients who were not referred to ABSCT, an allogeneic transplantation (TPL) was performed. Fifteen (21%) patients received palliative care or deceased, and their grafts were discarded in all but 1 patient. Additional grafts were discarded in 21 (31%) patients and stored in 9 (13%) patients who underwent ABSCT or allogeneic TPL (n = 44). Conclusions: As the role and efficacy of autologous HDCT/ABSCT are not established in the analyzed entities, the indication for PBSC collection should be reanalyzed in regular intervals. Moreover, PBSC grafts from patients who have deceased, have insufficient grafts, or have already undergone an allogeneic TPL should be considered for disposal or (if applicable) for research use, to economize storage costs on a rational basis.

Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B-cell lymphoma or acute lymphoblastic leukemia. Impaired T-cell fitness of CLL patients may be involved in treatment failure. Less-differentiated naive-like T cells play an important role in CART expansion and long-term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B-cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin-2-inducible T-cell kinase (ITK) which is involved in T-cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T-cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient-derived CART cells. Furthermore, ibrutinib enriched CART cells with less-differentiated naive-like phenotype and decreased expression of exhaustion markers including PD-1, TIM-3 and LAG-3. In addition, ibrutinib increased the cytokine release capacity of CLL patient-derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient-derived CART cell products.

Background: Modified immune cells (MICs) are mononuclear cells that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C. We recently showed that syngeneic MIC therapy controlled experimental autoimmune encephalitis (EAE) (1). In addition, allogeneic MIC therapy prevented rejection in rat heart and hindlimb as well as pig kidney transplantation (2). Objectives: We now wanted to translate these encouraging findings to the prevention and treatment of lupus nephritis. Methods: Splenocytes of syngeneic NZB/W F1 (BWF1) donor mice were incubated with mitomycin C and injected into recipient’s tail vein after matching for age and disease activity. Group 1 received no MIC therapy, group 2 standard-dose MIC therapy with 1.5x108/kg BW once and group 3 repeated MIC therapy with 1.5x108/kg BW at weeks 1, 2 and 3. Group 4 received MIC infusions before disease onset as preemptive treatment approach. Disease activity was monitored by loss of body weight, protein excretion, serum creatinine and dsDNA antibodies. Combined primary endpoint was day 40 post-treatment, protein excretion ≥3g/l for 2 consecutive weeks and >30% loss of original body weight. Histopathology with PAS and HE staining was performed to assess degree of lupus nephritis. Regulatory cell subsets were measured in peripheral blood. Results: MIC therapy prevented the progression of fatal lupus nephritis in BWF1 mice. Protein excretion, serum creatinine and dsDNA antibodies were lower in standard-dose (group 2) and preemptive (group 4) groups compared to control group (group 1) whereas repeated MIC therapy after disease onset had no effect (Figure 1A-E). The primary endpoint was reached significantly more often in control group (group 1, 67%) compared to treatment groups 2 (14%), 3 (14%) and 4 (0%) (Figure 1F). Renal architecture was preserved in different MIC treatment groups (groups 2-4) with decreased glomerular and tubular damage scores (Figure 1G). Most importantly, frequencies of CD8+CD25+FoxP3+ regulatory T cells and CD19+CD5+CD1dhigh regulatory B cells were significantly higher in MIC-treated (groups 2-4) compared to control animals of group 1, whereas double negative T cells were markedly reduced (Figure 1H). Conclusion: MIC therapy inhibits progression of active lupus nephritis. Interestingly, preemptive MIC therapy was even able to prevent onset of disease with no significant disease activity at completion of the study. In accordance with our previous pre-clinical EAE experiments (1) and a first in-human clinical trial in living-donor kidney transplantation (TOL-1 study), MIC therapy was able to induce an in-vivo induction of regulatory cell subsets. This clinically applicable cell therapeutic approach may control lupus nephritis by specifically silencing deleterious autoimmune responses. References: [1] Terness P, Oelert T, et al. Mitomycin C-treated dendritic cells inactivate autoreactive T cells: toward the development of a tolerogenic vaccine in autoimmune diseases. Proc Natl Acad Sci USA. 2008 Nov 25;105(47):18442–7. [2] Jiga LP, Ehser S, Kleist C, Opelz G, Terness P. Inhibition of heart allograft rejection with mitomycin C-treated donor dendritic cells. Transplantation. 2007 Feb 15;83(3):347–50. Disclosure of Interests: Claudius Speer Grant/research support from: Research Grant from TolerogenixX GmbH, Daniela Kim Grant/research support from: Research Grant from TolerogenixX GmbH, Christian Kleist Shareholder of: TolerogenixX GmbH, Anita Schmitt Shareholder of: TolerogenixX GmbH, Michael Schmitt: None declared, Claudia Sommerer: None declared, Andrea Steinborn: None declared, Florian Kalble: None declared, Christian Nusshag: None declared, Lei Wang Grant/research support from: Research Grant from TolerogenixX GmbH, Employee of: TolerogenixX GmbH, Alexander Kunz Grant/research support from: Research Grant from TolerogenixX GmbH, Employee of: TolerogenixX GmbH, Hanns-Martin Lorenz: None declared, Martin Zeier: None declared, Christian Morath Shareholder of: TolerogenixX GmbH, Matthias Schaier Shareholder of: TolerogenixX GmbH

IntroductionChimeric antigen receptor (CAR) T cells spark hope for patients with CD19+ B cell neoplasia, including relapsed or refractory (r/r) acute lymphoblastic leukaemia (ALL) or r/r non-Hodgkin’s lymphoma (NHL). Published studies have mostly used second-generation CARs with 4-1BB or CD28 as costimulatory domains. Preclinical results of third-generation CARs incorporating both elements have shown superiority concerning longevity and proliferation. The University Hospital of Heidelberg is the first institution to run an investigator-initiated trial (IIT) CAR T cell trial (Heidelberg Chimeric Antigen Receptor T cell Trial number 1 [HD-CAR-1]) in Germany with third-generation CD19-directed CAR T cells.Methods and analysisAdult patients with r/r ALL (stratum I), r/r NHL including chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma (stratum II) as well as paediatric patients with r/r ALL (stratum III) will be treated with autologous T-lymphocytes transduced by third-generation RV-SFG.CD19.CD28.4-1BB zeta retroviral vector (CD19.CAR T cells). The main purpose of this study is to evaluate safety and feasibility of escalating CD19.CAR T cell doses (1–20×106transduced cells/m2) after lymphodepletion with fludarabine (flu) and cyclophosphamide (cyc). Patients will be monitored for cytokine release syndrome (CRS), neurotoxicity, i.e. CAR-T-cell-related encephalopathy syndrome (CRES) and/or other toxicities (primary objectives). Secondary objectives include evaluation ofin vivofunction and survival of CD19.CAR T cells and assessment of CD19.CAR T cell antitumour efficacy.HD-CAR-1 as a prospective, monocentric trial aims to make CAR T cell therapy accessible to patients in Europe. Currently, HD-CAR-1 is the first and only CAR T cell IIT in Germany. A third-generation Good Manufacturing Practice (GMP) grade retroviral vector, a broad spectrum of NHL, treatment of paediatric and adult ALL patients and inclusion of patients even after allogeneic stem cell transplantation (alloSCT) make this trial unique.Ethics and disseminationEthical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings.Trial registration numberEudra CT 2016-004808-60;NCT03676504; Pre-results.

Background Modified immune cells (MICs) are mononuclear cells that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C. We showed that syngeneic MIC cell therapy prevented experimental autoimmune encephalitis (EAE) as well as alloreactive rejection in rat heart and hindlimb and pig kidney transplantation. We now aimed to translate these encouraging investigations to prevent murine lupus nephritis and control autoimmunity by MIC therapy. Methods Splenocytes of syngeneic NZB/W F1 (BWF1) mice were harvested from a donor, incubated with mitomycin C and injected into recipients tail vein after matching for age and disease activity. Group 1 received no MIC therapy, group 2 standard-dose MIC therapy with 1.5 × 108/kg BW once and group 3 repeated MIC therapy with 1.5 × 108/kg BW at week 1, 2 and 3, respectively. Group 4 was dosed with repeated MIC therapy before disease onset as preemptive treatment approach. Disease activity was monitored by loss of body weight, protein excretion and serum creatinine. Combined primary endpoint was day 40 post-treatment, protein excretion 3 g/L for 2 consecutive weeks and >30% loss of original body weight. Histopathology with PAS and HE staining was performed to assess degree of lupus nephritis. In the peripheral blood we determined regulatory cell subsets. Results MIC therapy prevented the progression of fatal lupus nephritis in BMF1 mice. Protein excretion and serum creatinine were lower in standard-dose group and preemptive group compared to control group whereas repeated MIC therapy in active disease failed to inhibit both significantly. Accomplishment of combined primary endpoint was significantly increased in group 1 (67%) compared to group 2 (14%), group 3 (14%) and group 4 (0%), respectively. Renal architecture was preserved throughout different MIC treatment groups with decreased glomerular and tubular damage scores. After MIC therapy, frequencies of CD8 +CD25+FoxP3+regulatory T cells and CD19 +CD5+CD1 dhigh regulatory B cells increased significantly, whereas double negative T cells were markedly reduced throughout all treatment groups. Conclusions MIC therapy inhibits progression of active lupus nephritis. Interestingly, preemptive MIC therapy was even able to prevent onset of disease since no significant disease activity was observed at completion of the study. In accordance with our previous pre-clinical EAE experiments and a first in-human clinical trial in living-donor kidney transplantation (TOL-1 study), MIC therapy enables an in-vivo induction of regulatory cell subsets. This clinically applicable cell therapeutic approach may control lupus nephritis by specifically silencing deleterious autoimmune response. Funding Source(s): Research grant from TolerogenixX GmbH Disease activity was assessed after MIC cell therapy by loss of original body weight (A), protein excretion via semiquantitative sticks (B), albuminuria via ELISA (C) and serum creatinine via ELISA (D). Primary endpoint was defined as day 40 post-treatment, protein excretion 3 g/L for 2 consecutive weeks and >30% loss of original body weight (E). Histopathology with PAS and HE staining was performed. Representative PAS staining for different treatment groups and the control group are shown (F). Different regulatory cell subsets as well as DN T cells were determined in peripheral blood. Representative dot plots of regulatory CD19 +CD5+CD1 dhigh B cells, CD8 +CD25+FoxP3+T cells and CD3 +CD4 CD8- double-negative T cells are shown (G).

Refinement of immunosuppressive strategies has led to further improvement of kidney graft survival in recent years. Currently, the main limitations to long-term graft survival are life-threatening side effects of immunosuppression and chronic allograft injury, emphasizing the need for innovative immunosuppressive regimens that resolve this therapeutic dilemma. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness have been tested in early phase I and phase II clinical trials in kidney transplantation. The aim of this overview is to summarize current cell therapeutic approaches to immunosuppression in clinical kidney transplantation with a focus on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). MICs show great promise as a therapeutic agent to achieve the rapid and durable establishment of donor-unresponsiveness in living-donor kidney transplantation. Cell-based therapeutic approaches may eventually revolutionize immunosuppression in kidney transplantation in the near future.

// Michael Schmitt 1, * , Angela G. Huckelhoven 1, * , Michael Hundemer 1 , Anita Schmitt 1 , Susanne Lipp 1 , Martina Emde 1 , Hans Salwender 2 , Mathias Hanel 3 , Katja Weisel 4 , Uta Bertsch 1 , Jan Durig 5 , Anthony D. Ho 1 , Igor Wolfgang Blau 6 , Hartmut Goldschmidt 1, 7 , Anja Seckinger 1 and Dirk Hose 1 1 Universitatsklinikum Heidelberg, Medizinische Klinik V, Heidelberg, Germany 2 Department of Internal Medicine II, Asklepios Klinik Altona, Hamburg, Germany 3 Department of Internal Medicine III, Klinikum Chemnitz GmbH, Chemnitz, Germany 4 Department of Hematology, Oncology and Immunology, University of Tubingen, Tubingen, Germany 5 Department of Hematology, University Hospital Essen, Essen, Germany 6 Medical Clinic III Hematology and Oncology, Charite University Medicine Berlin, Berlin, Germany 7 Nationales Centrum fur Tumorerkrankungen, Heidelberg, Germany * These authors contributed equally to this work Correspondence to: Dirk Hose, email: dirk.hose@med.uni-heidelberg.de Keywords: tumor associated antigens, T cells, immunogenicity, multiple myeloma, RNA-sequencing Received: May 20, 2016 Accepted: July 13, 2016 Published: August 11, 2016 ABSTRACT Background: Raising T-cell response against antigens either expressed on normal and malignant plasma cells (e.g. HM1.24) or aberrantly on myeloma cells only (e.g. cancer testis antigens, CTA) by vaccination is a potential treatment approach for multiple myeloma. Results: Expression by GEP is found for HM1.24 in all, HMMR in 318/458 (69.4%), MAGE-A3 in 209/458 (45.6%), NY-ESO-1/2 in 40/458 (8.7%), and WT-1 in 4/458 (0.8%) of samples with the pattern being confirmed by RNA-sequencing. T-cell-activation is found in 9/26 (34.6%) of patient samples, i.e. against HM1.24 (4/24), RHAMM-R3 (3/26), RHAMM1-8 (2/14), WT-1 (1/11), NY-ESO-1/2 (1/9), and MAGE-A3 (2/8). In 7/19 T-cell activation responses, myeloma cells lack respective antigen-expression. Expression of MAGE-A3 , HMMR and NY-ESO-1/2 is associated with adverse survival. Experimental design: We assessed expression of HM1.24 and the CTAs MAGE-A3 , NY-ESO-1/2 , WT-1 and HMMR in CD138-purified myeloma cell samples of previously untreated myeloma patients in the GMMG-MM5 multicenter-trial by gene expression profiling (GEP; n = 458) and RNA-sequencing ( n = 152) as potential population regarding vaccination trials. We then validated the feasibility to generate T-cell responses ( n = 72) against these antigens by IFN-γ EliSpot-assay ( n = 26) related to antigen expression ( n = 22). Lastly, we assessed survival impact of antigen expression in an independent cohort of 247 patients treated by high-dose therapy and autologous stem cell transplantation. Conclusions: As T-cell responses can only be raised in a subfraction of patients despite antigen expression, and the number of responses increases with more antigens used, vaccination strategies should assess patients’ antigen expression and use a “cocktail” of peptide vaccines.

Dendritic cells (DCs) are the most professional antigen-presenting cells eliciting cellular and humoral immune responses against cancer cells by expressing these antigens on MHC class I/II complexes to T cells. Therefore, they have been employed in many clinical trials as cancer vaccines for patients with cancer. This review focuses on the use of DCs in leukemia patients expressing leukemia-associated antigens (LAAs).The contribution of both stimulating vs. tolerogenic DCs as well as of other factors to the milieu of anti-leukemia immune responses are discussed. Several DC vaccination strategies like leukemia lysate, proteins and peptides have been developed. Next generation DC vaccines comprise transduction of DCs with retroviral vectors encoding for LAAs, cytokines and costimulatory molecules as well as transfection of DCs with naked RNA encoding for LAAs. Published as well as ongoing clinical trials are reported and critically reviewed.Future results will demonstrate whether next-generation DCs are really superior to conventional pulsing with peptide, protein or tumor lysate. However, currently available methods based on nucleic acid transfection/transduction are tempting in terms of material production costs and time for clinical application according to good manufacturing practice (GMP).

Terumo BCT recently introduced a new system for mononuclear cell (MNC) collection that uses a Spectra Optia apheresis machine equipped with a redesigned disposable kit and software program (version 11.2). It allows for the continuous collection of MNCs, unlike the original Spectra Optia system (version 7.2), which included a chamber for two-step cell separation. The aim of this study was to compare the two apheresis systems in regard to specific performance parameters. A retrospective data analysis of 150 patients who had undergone peripheral blood stem cell collection between March of 2014 and May of 2015 at our institution was performed. For the matched comparison, patients were divided into two groups by diagnosis and by previous forms of therapy received: a homogeneous group of patients with multiple myeloma (MM) that had received first line therapy ("MM" group, n = 88) and a heterogeneous group that included all of the other patients ("other" group, n = 62). No significant differences in CD34+ collection yields between both collection regimens were found (pMM = 0.19, pother = 0.74) in either group. Moreover, similar performance ratios (collected/predicted CD34+ cell number in %) were observed (pMM = 0.89, pother = 0.1). No relevant variations in platelet or hemoglobin loss were found between the two systems. We conclude that the new continuous Spectra Optia MNC system is equally efficient in collecting CD34+ cells and can be used without sacrificing collection efficiency levels when treating a broad variety of autologous patients. J. Clin. Apheresis 32:27-34, 2017. © 2016 Wiley Periodicals, Inc.

Introduction: Cytomegalovirus (CMV) reactivation occurs in seronegative patients after solid organ transplantation (SOT) particularly from seropositive donors and can be lethal. Generation of CMV-specific T cells helps to prevent CMV reactivation. Therefore, we initiated a clinical phase I CMVpp65 peptide vaccination trial for seronegative end-stage renal disease patients waiting for kidney transplantation. Methods: The highly immunogenic nonamer peptide NLVPMVATV derived from CMV phosphoprotein 65(CMVpp65) in a water-in-oil emulsion (Montanide™) plus imiquimod (Aldara™) as an adjuvant was administered subcutaneously four times biweekly. Clinical course as well as immunological responses were monitored using IFN-γ ELISpot assays and flow cytometry for CMV-specific CD8+ T cells. Results: Peptide vaccination was well tolerated, and no drug-related serious adverse events were detected except for Grade I–II local skin reactions. Five of the 10 patients (50%) mounted any immune response (responders) and 40% of the patients presented CMV-specific CD8+ T cell responses elicited by these prophylactic vaccinations. No responders experienced CMV reactivation in the 18 months post-transplantation, while all non-responders reactivated. Conclusion: CMVpp65 peptide vaccination was safe, well tolerated, and clinically encouraging in seronegative end-stage renal disease patients waiting for kidney transplantation. Further studies with larger patient cohorts are planned.

Many transplantation centers routinely collect 1 or more autologous peripheral blood stem cell (PBSC) grafts in patients with hemato-oncologic and autoimmune disorders. However, subsequent high-dose chemotherapy and autologous blood stem cell transplantation (ABSCT) are often not performed, for various reasons. Currently, little is known about the actual utilization rate of stored PBSCs. We retrospectively analyzed the collection, storage, and disposal practices of PBSC products from a large cohort of patients (n = 1020) with hematologic, oncologic, and autoimmune disorders at our institution over a 12-year period. Patients with multiple myeloma were excluded. Based on our institution-specific charges, we estimated the costs for PBSC collection/processing and storage. The median number of sufficient PBSC collections per patient in the whole cohort was 2 (range, 1 to 6). We could demonstrate that only 67% of all patients who had collected sufficient PBSCs for transplantation actually underwent ABSCT, and only a small minority of all patients (4%) underwent multiple ABSCTs. The actual use of the stored PBSC grafts varied among disease entities from >80% to 0%. From a retrospective standpoint, the collected and discarded (definitively not used) or stored (potentially not used) cryostored PBSCs were associated with considerable costs of collection, cryopreservation, and long-term cryostorage. Although keeping open the therapeutic option for future transplantations may be important, there is currently a huge discrepancy between collection/storage practices and actual utilization of the cryopreserved PBSCs, at a considerable cost and strain on patients. Our study provides a rationale for reevaluating the present standards.

Wilms' tumor gene 1 (WT1) is overexpressed in leukemia and WT1-derived CD8(+) T-cell epitopes for immunotherapies targeting WT1 have been defined. Here, we analyzed expression of WT1 in 226 peripheral blood and bone marrow samples from patients with acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) before and after allogeneic stem cell transplantation (SCT). Transcripts were assessed by quantitative polymerase chain reaction, and WT1-specific CD8+ cytotoxic T cells (CTL) were monitored by tetramer staining and enzyme-linked immunospot (ELISPOT) assays. Reduction of WT1 levels correlated with a longer survival (p

Successful collection of peripheral blood stem cells (PBSCs) depends on the optimal orchestration of mobilization chemotherapy, granulocyte colony stimulating factor (G-CSF) application, and CD34+ cell number assessment in the peripheral blood (PB). However, determining the optimal timing in accordance to the applied chemomobilization regimen can be challenging. Although most centers apply their own local timing schedules, a reliable timetable including the currently most often used mobilization regimens is lacking. We present a comprehensive analysis of the timing modalities for 11 of the most commonly used chemomobilization regimens. A retrospective analysis was performed on the clinical and PBSC collection parameters (including duration of G-CSF application, time point of CD34+ assessment, PB CD34+ cell count, number of leukapheresis [LP] sessions, processed blood volume, and CD34+ collection results) of 91 representatively selected patients who had undergone stem cell mobilization at 2 collection centers. Six to 10 patients were analyzed per regimen with a variety of diagnoses, including multiple myeloma, malignant lymphoma, and sarcoma. No collection failures ( The presented data on the timing of chemomobilization, G-CSF application, and stem cell collection may be helpful in clinical decision making and contribute to a more transparent and predictable treatment process.

Despite high-dose chemotherapy followed by autologs stem-cell transplantation as well as novel therapeutic agents, multiple myeloma (MM) remains incurable. Following the general trend towards personalized therapy, targeted immunotherapy as a new approach in the therapy of MM has emerged. Better progression-free survival and overall survival after tandem autologs/ allogeneic stem cell transplantation suggest a graft versus myeloma effect strongly supporting the usefulness of immunological therapies for MM patients. How to induce a powerful antimyeloma effect is the key issue in this field. Pivotal is the definition of appropriate tumor antigen targets and effective methods for expansion of T cells with clinical activity. Besides a comprehensive list of tumor antigens for T cell-based approaches, eight promising antigens, CS1, Dickkopf-1, HM1.24, Human telomerase reverse transcriptase, MAGE-A3, New York Esophageal-1, Receptor of hyaluronic acid mediated motility and Wilms’ tumor gene 1, are described in detail to provide a background for potential clinical use. Results from both closed and ongoing clinical trials are summarized in this review. On the basis of the preclinical and clinical data, we elaborate on three encouraging therapeutic options, vaccine-enhanced donor lymphocyte infusion, chimeric antigen receptors–transfected T cells as well as vaccines with multiple antigen peptides, to pave the way towards clinically significant immune responses against MM.

Background Harvest of more than one CD34+ stem cell transplant has become the standard, to ensure the option for a second autologous transplantation in patients with relapsed or progressive multiple myeloma (MM). Additional administration of the CXCR-4 inhibitor plerixafor has been shown to increase the efficiency of CD34+ stem cell harvest. However, the algorithm when to apply plerixafor is still under debate. Study Design and Methods In this retrospective study, 46 MM patients were categorized into four groups according to their CD34+ stem cell count in peripheral blood (PB) and mobilization with or without plerixafor: Group A comprised poor mobilizers with CD34+ cell counts of fewer than 20 × 106/L in PB. Group B included inadequate mobilizers with CD34+ cell counts of 20 × 106/L or more in PB and a low CD34+ stem cell yield in the first leukapheresis session. Patients receiving plerixafor preemptively (Group A1) and as a rescue strategy (Group B1) were compared to patients continuing stem cell collection with granulocyte–colony-stimulating factor alone (Groups A2 and B2). Results In both, the preemptive and the rescue settings, plerixafor enhanced the CD34+ stem cell yield significantly. Poor mobilization and administration of plerixafor was not associated with delayed engraftment. Conclusion Our data demonstrate that administration of plerixafor is safe and effective and facilitates a significantly higher CD34+ stem cell harvest. Based on the presented data, we propose an algorithm for the use of plerixafor for CD34+ stem cell mobilization and harvesting in poor mobilizing myeloma patients.

While extensive data demonstrated that plerixafor improves stem cell harvest in difficult-to-mobilize patients, economic concerns limit a broader application. We retrospectively assessed the effect of an early plerixafor rescue regimen for mobilization in patients with multiple myeloma. Patients were intended for high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ABSCT) and therefore received cyclophosphamide-based mobilization chemotherapy and consecutive stimulation with granulocyte colony-stimulating factor (G-CSF). Fifteen patients with poor stem cell harvest in the first leukapheresis session received plerixafor. Data were compared with a matched historic control group of 45 patients who also had a poor stem cell yield in the first apheresis session, but continued mobilization with G-CSF alone. Patients in the plerixafor group collected significantly more CD34+ cells in total (median 4.9 vs. 3.7 [range 1.6–14.1 vs. 1.1–8.0] × 106 CD34+ cells /kg bw; P

Background: Chimeric antigen receptor (CAR) engineered T cell therapy is currently revolutionizing the field of cancer immunotherapy. However, further enhancement of the function of CAR T cells either through modification of CAR structure or through combination with other therapies is intensively investigated. Resistance to apoptosis is one of the hallmarks of cancer biology. Overexpression of anti-apoptotic proteins of the Bcl-2 family proteins, such as Bcl-2, Mcl-1 and Bcl-xl is considered primarily responsible for the increased apoptosis resistance and prolonged survival of hematological tumor cells. Apoptosis inhibitor blockade agents (AIBAs) such as Bcl-2, Mcl-1 and COX inhibitors could sensitize malignant cells to apoptosis. Therefore, combining CAR T cell therapy with AIBAs might be a promising approach to increase the therapeutic response. However, the third generation of CAR T cells including a 4-1BB co-stimulatory domain which triggers a signaling cascade with the upregulation of anti-apoptotic molecules might be hampered by AIBAs. Therefore, in this study we analyzed the influence of AIBAs on third generation CD19 CAR T cells. Materials and methods: CD19 CAR T cells were manufactured using a 3rd generation CAR vector containing both CD28 and 4-1BB co-stimulatory domains. The expression of anti-apoptotic Bcl-2 family members in leukemia/lymphoma cell lines and in CD19 CAR T cells have been assessed by quantitative real time polymerase chain reaction (qRT-PCR), western blot (WB) and flow cytometery. The optimal concentrations of inhibitors have been determined by CellTiter Glo™ assay. The effect of inhibitors on CAR T cells and tumor cells has been evaluated by chromium-51 (51Cr) release assay, Calcein AM™ assay and FACS-based apoptosis assay. Intracellular cytokine staining and surface marker staining were performed to determine the function of CAR T cells. Results: qRT-PCR and WB showed that leukemia/lymphoma cell lines 380 and U698M had the highest Bcl-2 (qRT-PCR: 1.73 ± 0.04, p = 0.001; WB: 1.82 ± 0.54, p = 0.016) and Mcl-1 (qRT-PCR: 12.39 ± 1.37, p = 0.000; WB: 1.92 ± 1.08, p = 0.142) expression levels, respectively,. These findings were confirmed by anti-apoptotic BCL-2 family protein intracellular staining. Elevated protein expression of Mcl-1, Bcl-2 and Bcl-xl was observed in CAR T cells upon 380 cell stimulation. The manufactured CD19 CAR T cells exhibited specific killing on CD19+ tumor cells. A slightly enhanced killing efficiency of CAR T cells was observed when 380 cells were co-cultured with CAR T cells in the presence of ABT199 (0 µM vs. 1 µM: 54.21 ± 3.23 vs. 69.06 ± 2.17, p = 0.0041). This result could be explained by a higher sensitivity of 380 cells to ABT199 than CART cells and by an increased activation of CAR T cells. Even though the killing efficiency of CAR T cells was not improved by S63845, the CD107a+TNF-α+IFN-γ+ CAR T cells were increased as well as the proliferation and persistence of CAR T cells were strengthened. Of note, pretreatment of tumor cells with inhibitors could significantly enhance the killing efficiency of CAR T cells via upregulation of CD19 antigen on tumor cells (ABT199-pretreated 380 cells: 0 µM vs.1 µM, 42.59 ± 5.82 vs. 54.58 ± 4.87, p = 0.0493; S63845-pretreated-U698M cells: 0 µM vs. 0.3 µM, 31.88 ± 4.77 vs. 66.35 ± 8.09, p = 0.000). Conclusion: The quantity of CAR T cells can be negatively affected by Apoptosis inhibitor blockade agents. However, the quality of CAR T cells could be improved. The cytotoxic effect of CAR T cells can be further enhanced by pretreatment of tumor cells with inhibitors. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support.

Introduction T cells transduced with a chimeric antigen receptor (CAR) have demonstrated significant clinical efficacy in patients with lymphoid malignancies including relapsed or refractory (r/r) B-lineage acute lymphoblastic leukemia (ALL) or r/r B-cell non-Hodgkin's lymphoma (NHL). Second-generation CAR T cells comprising 4-1BB or CD28 as costimulatory domains have become commercially available for the treatment of patients with CD19+ lymphoid malignancies. However, achievement of durable clinical responses remains a challenge in CAR T cell therapy. Consequently, third-generation CARs incorporating both elements might display short-term efficacy with potent and rapid tumor elimination (CD28) as well as long-term persistence (4-1BB). So far, only two clinical trials employing third-generation CAR T cells have been reported. Both enrolled 31 patients in summary and demonstrated favorable results for third-generation CAR T cells. Here, we report on first results of our investigator-initiated trial (IIT) on third-generation CD19-directed CAR T cells: The Heidelberg CAR trial 1 (HD-CAR-1; NCT03676504; EudraCT 2016-004808-60) is a phase I/II trial initiated in September 2018 with in-house leukapheresis and CAR T cell manufacturing in full compliance with European Good Manufacturing Practice (GMP) guidelines at the University Hospital Heidelberg. Methods Adult and pediatric patients with r/r ALL and patients with r/r chronic lymphocytic leukemia (CLL) or NHL including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL) are treated with autologous T lymphocytes transduced with a CD19 targeting third-generation CAR retroviral vector (RV-SFG.CD19.CD28.4-1BBzeta). The main purpose of HD-CAR-1 is to evaluate safety and feasibility of escalating third-generation CAR T cell doses (1-20×106 transduced cells/m2) after lymphodepletion with fludarabine (30 mg/m2/d on days -4 to -2) cyclophosphamide (500 mg/m2/d on days -4 to -2). Patients are monitored for cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and/or other toxicities. In vivo function, survival and anti-tumor efficacy of CAR T cells are assessed. Results To date, 10 patients (3 adult ALL, 2 CLL, 2 MCL, 2 DLBCL, 1 transformed FL) have been enrolled and subjected to leukapheresis. Transduction efficiency of T lymphocytes ranged between 33%-66% and high numbers of transduced CAR T cells were harvested (70-123x106 CAR T cells). No production failure occurred. CAR T cell products were sterile and free from mycoplasma and endotoxins. The copy number per CAR T cell did not exceed 7. Eight patients (2 adult ALL, 2 CLL, 1 MCL, 2 DLBCL, 1 transformed FL) have received the CAR T cell product (6 patients: 106 transduced cells/m2; 2 patients 5×106 transduced cells/m2). No signs of CRS or ICANS > grade 2 have been observed. Only one patient required tocilizumab. No neurological side-effects occurred, even not in patients with involvement of the central nervous system (CNS). In quantitative real-time PCR, CAR T cells were detectable in the peripheral blood (PB) in 3 of 4 analyzed patients or the cerebrospinal fluid (CSF) of an ALL patient with CNS involvement. The CAR T cell frequency reached up to 200,000 copies/µg DNA, in some patients beyond end-of-study at day 90 after CAR T cell administration. Clinical responses to treatment were observed in 6/8 (75%) treated patients so far (2/8 patients have received CAR T cells recently and are not yet evaluable for response). Conclusion Leukapheresis and CAR T cell manufacturing were effective for all patients enrolled in the HD-CAR trial to date. Patients responded clinically to treatment despite low numbers of administered CAR T cells. CAR T cells displayed an excellent safety profile and were detectable for more than 3 months following administration. Furthermore, CAR T cells migrated into different compartments including the CSF in case of CNS involvement. For HD-CAR-1 leukapheresis, CAR T cell manufacturing, CAR T cell administration, patient monitoring and follow-up are performed in-house, providing autarky from transport or production sites outside the University Hospital Heidelberg. Altogether, HD-CAR-1 accounts to clinical evaluation of third-generation CAR T cells that might contribute to long-term CAR T cell persistence, hence improving efficient and durable responses in treated patients. Disclosures Schmitt: Therakos Mallinckrodt: Other: Financial Support . Sellner:Takeda: Employment. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:Therakos Mallinckrodt: Other: Financial Support; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia.

Introduction: Systemic steroids with their strong immunosuppressive and anti-inflammatory effects are the current gold standard for initial treatment of the graft-versus-host disease (GvHD). But, the effectiveness of high-dose steroids comes along with an increased susceptibility to infections and other secondary malignancies. Extracorporeal photophoresis (ECP), an immunotherapeutic therapy, is currently being used as a clinically well-established second-line treatment for GvHD. Contrary to steroid treatment, there is no clinical data showing that ECP is associated with an increased risk of infection. However, the exact mechanism of action how ECP preserves the anti-viral and anti-leukemic effects on the cellular level has not been discovered yet. Materials and methods: Thirty-four patients with steroid-refractory/resistant aGvHD ≥ II and moderate to severe cGvHD were treated with ECP at the University Hospitals in Heidelberg, Greifswald in Germany and Chaim Sheba Medical Center in Israel. A comprehensive analysis of cell subsets was performed using multi-parametric flow cytometry. Additionally, the quantity and quality of CMV-specific T cells was determined by tetramer staining and interferon-γ enzyme-linked immunospot assay. The NK activity in terms of killing functionality and cytokine release was analyzed by intracellular cytokine staining and chromium-51 release assay, respectively. The proliferative capacity of effector cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Results: ECP therapy was effective with an overall response rate of 75% for patients with aGvHD (x/y) and 78% (x/y) for patients with cGvHD. Of note, all patients showed neither the increased susceptibility to infections nor the reactivation of CMV nor the tumor relapse. Overall, the frequency of well-established protective cell subsets in terms of cytotoxic CD8+ T cells, CD4+CD8+ T cells, γδ T cells, NK cells and NKT cells remained constant under the ECP therapy. Specifically, no significant influence of ECP therapy on the CD56dimCD57+NKG2C+ NK cells, a specialized anti-viral/tumor population, and the CMV+CD8+ T cells could be observed. The further phenotyping of CMV+CD8+ T cells showed that CD45RA+CCR7-CMV+CD8+ TE cells and CD45RA-CCR7-CMV+CD8+ TEM cells could keep stable under the ECP therapy. Besides these, priming, differentiation and maturation of NK cells through upregulation of CD57 by ECP therapy bridged the T-cell-deficient period after HSCT in order to control the viral infections and eliminate the residual malignant cells. Moreover, ECP could reduce the CD62L expression on TE population which not only facilitates the hematopoietic engraftment and contributes to the phenotypic and functional T cell reconstitution after transplantation without causing GvHD, but also enhances the functional immune reconstitution against tumor and viral antigens. Functionally, neither IFN-g released by virus specific T cells nor production of TNF-a and IFN-g by NK cells in response to K562 cells was hampered by ECP. Of note, the functions of NK cells in terms of the MFI of cytokines and the polyfunctionality of NK cells were stable under ECP treatment. Additionally, the proliferation of NK cells, CD4+ T cells and CD8+ T cells providing an expanded pool of effector cells against the pathogens was not hampered by ECP therapy as well. Conclusions: ECP therapy constitutes an effective strategy to treat GvHD. In addition, ECP appears to be a safe therapy compared to continued steroid use. Our current results suggest that ECP allows to maintain immunity against infections and tumors. Disclosures Schönland: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Dreger:Neovii, Riemser: Research Funding; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support .

Novel therapies with chimeric antigen receptor (CAR)-transduced T cells (TCs) sparked new hope for patients with relapsed or refractory CD19-positive leukemia or lymphoma even after stem cell therapies. This review focuses on CARs recognizing the B cell antigen CD19. Both retroviral and lentiviral vectors are used, encoding various anti-CD19 CAR constructs comprising costimulatory molecules such as CD28, CD137/4-1BB, and OX40 either alone (second-generation CARs) or in combination (third-generation CARs). Current, up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 58 ongoing trials is given. Clinical responses were achieved in up to 81% of ALL, 50% of CLL, and 40% of NHL patients. Factors with potential influence on the clinical outcome might be the design of the vector, the preconditioning regimen, and the number and quality of transfused CAR TCs. The applicability of clinical CAR TC therapy might include relapse after allogeneic stem cell transplantation (alloSCT), and ineligibility for or "bridging" until alloSCT. In summary, CAR therapy represents a highly promising treatment option even in heavily pretreated patients.

The favorable graft-versus-leukemia (GVL) effect that occurs after stem cell transplantation suggests that T cells can eradicate leukemia blasts. T cells specifically recognize peptides and exert an antileukemia effect. Several leukemia-associated antigens (LAAs) have been found to be overexpressed in malignant cells from patients with acute or chronic leukemia leading to the generation of peptide-based leukemia immunotherapy. Peptide vaccination with LAAs, whose expression is low in normal tissue, such as the receptor for hyaluronic acid-mediated motility (RHAMM), Wilms tumor 1 (WT1), proteinase-3 (PR-3) and the breakpoint cluster region-Abelson (bcr-abl) has been reported to induce leukemia-specific T-cell responses in patients with a variety of hematological malignancies. Moreover, the immune responses achieved after vaccination positively correlated with good clinical outcomes, that is complete remission. Large efforts have been made to optimize the dose and format of vaccines, as well as their adjuvants, in small pilot trials. In this article we summarize clinical Phase I and II peptide vaccination trials with RHAMM, WT1, PR-3 and bcr-abl for leukemia patients.

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal. STUDY DESIGN AND METHODS: Here, the novel streptamer technology was used at good manufacturing practice (GMP) level for adoptive transfer of CMV-specific T cells into acute leukemia patients with recurrent high CMV antigenemia after allogeneic stem cell transplantation. RESULTS: After a single transfusion, the frequency of CMV-specific CD8+CD45RA+CCR7– effector T cells increased dramatically from 0.0% to a maximum of 27.1% of all T cells. These T cells were clearly donor derived and did not stem from intrinsic reconstitution, as demonstrated by analysis of 1) donor chimerism through single-tandem repeats, 2) T-cell receptor excision circles, and 3) Vβ-chain typing by polymerase chain reaction. Clinically, the specific T-cell transfer resulted in a persistent clearance of the CMV antigenemia, which allowed the patients to discontinue toxic antiviral drug therapy without further high-level reactivation of CMV, demonstrating the power of the streptamer technology. CONCLUSION: Taken together, the streptamer technology offers the advantage of selecting virus-specific CD8+ T cells at GMP level for adoptive T-cell transfer, thus inducing long-lasting specific CD8+ T-cell responses without increasing the risk for graft-versus-host disease.

Background: After transplantation of solid organs like allogeneic kidneys, the administration of immunosuppressive drugs such as cyclosporine A (CSA) and steroids is mandatory. This regimen exerts toxicity to the graft and makes transplant recipients prone to opportunistic infections. Replacement of the immunosuppressive drugs by a transfusion of tolerogenic cells might overcome these noxious side effects. Mitomycin-induced cells (MICs) are donor-derived monocytes that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C and have a myeloid-derived suppressor cell (MDSC) character. Materials and methods: Peripheral blood mononuclear cells (PBMCs) were harvested from living kidney donors by leukapheresis and MIC cells were manufactured under Good Manufacturing Practice (GMP) conditions in the clean room of our University Hospital. Kidney transplant recipients received either 1.5x10E6 MIC cells per kg body weight on day -2 (N=3, group A) or 1.5x10E8 MIC cells per kg body weight on day -2 (N=3, group B) or on day -7 (N=4, group C) before living donor kidney transplantation. Patients received immunosuppressive therapy with cyclosporine a (CSA), enteric coated mycophenolate sodium (EC-MPS) and corticosteroids. The primary outcome was measured by the frequency of adverse events (AEs) on post-transplant day 30 with a follow-up until post-transplant day 360 for all patients. Results: Clinically, all kidney transplant recipients showed a median serum creatinine of 1.4 mg/dL at day 30 and remained stable with a median creatinine of 1.48 mg/dL at day 180 without significant proteinuria (median 10 g/mol creatinine at day 180) and without rejection episode. In total 72 AEs were observed including three severe AEs which were not associated with the MIC cell transfusion. Besides two infectious complications, no positive cross match results, no de novo donor-specific antibodies or rejection episodes were recorded. In group C, a reduction of immunosuppressive therapy was effective in the observational phase with low-dose CSA and low-dose EC-MPS. Immunologically, CD19+ B cells increased up to a median of 300/µL until day 30, followed by a decrease to a median of 35/µL at day 180 in group C. Notably, CD19+CD24highCD38high regulatory B cells were significantly increased from a median of 2% on day 30 to a median of 20% on day 180. The plasma IL-10/TNF-α ratio increased from a median of 0.05 before cell therapy to a median of 0.11 at day 180. Moreover, recipient lymphocytes showed no or only minimal reactivity against irradiated donor PBMCs, while reactivity against 3rd party healthy donor PBMCs in vitro was not impaired. Additionally, the quality assessment demonstrated that MIC cells have the capability to induce tolerogenic dendritic cells (tDCs) by down-regulating the costimulatory molecules CD80 and CD86, and the maturation molecule CD83, while up-regulating the immunosuppressive molecule CD103. MIC-induced tDCs showed the capacity to inhibit donor specific allo-reactive CD4 and CD8 T cell proliferation. Conclusion: A stable function was observed in all transplant recipients receiving the MIC cells product without any allograft injury or rejection episodes even under reduction of conventional therapy with immunosuppressive drugs. MIC cells constitute a novel tool for immunotherapy with a high potential in transplantation medicine. Disclosures No relevant conflicts of interest to declare.

Introduction: Graft-versus-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), results in post-transplant morbidity and mortality. Steroids with strong immunosuppressive and anti-inflammatory effects remain the gold standard for initial management of GvHD. However, high doses of steroids are usually accompanied by an increased risk of infections and secondary malignancies. ECP, due to its good clinical response, is recommended as a second-line treatment for GvHD. Notably, no clinical data showed that ECP therapy results in relapse and infection. However, the mechanism of action behind that is barely known. Therefore, we conducted this study to figure out how ECP preserves the anti-viral and anti-leukemia effects. Materials and Methods: 34 patients with steroid-refractory/resistant aGvHD ≥ °II and moderate to severe cGvHD received ECP therapy at the University Hospitals Heidelberg, Greifswald and Tel Hashomer. ECP therapy was performed according to the current European guidelines. For clinical staging of aGvHD under ECP treatment patients were evaluated according to Glucksberg criteria, for quality of life according to Karnofsky and for clinical staging of cGvHD according to NIH criteria. Phenotypical analysis of different cellular subsets was evaluated by multicolor flow cytometry. The quantity and quality of virus-specific CD8+ T cells were evaluated by Tetramer staining and IFN-γ-ELISPOT. NK activity in terms of killing function and cytokine release function was monitored by chromium-51 release assay and intracellular cytokine staining. Results: ECP seems to be favorable in the treatment of GvHD. Clinically, an overall response of 75% for aGvHD and 78% for cGvHD patients was achieved. A steroid-sparing effect in addition to the resolution of GvHD manifestations was observed in responders. Our patients showed no increased susceptibility to infections. On the cellular level, the frequency of cytotoxic CD8+ T cells, the most important mediators of GvL activity, as well as CD4+CD8+ T cells, γδ T cells, NKT cells and CD56dimCD57+NKG2C+ NK cells as other well-established protective cell subsets remained constant under ECP therapy. Moreover, neither frequency nor IFN-γ release of CMV specific CD8+ T cells was hampered by ECP. 51Cr-release assay revealed stable functional cytotoxicity of NK cells. Additionally, ECP therapy did not affect the capacity of cytokine release by NK cells, including quality, quantity and multifunctional release. Furthermore, no significant influence of ECP therapy on antigen recall function of NK cells, CD4+ and CD8+ T cells could be observed. Conclusion: ECP therapy represents an attractive strategy to treat GvHD. Moreover, our results reflect that ECP therapy preserves immunity against infections as well as the GvL effect via maintaining the quality and quantity of effector cells. Disclosures Hilgendorf: Novartis: Other: Travel support, Research Funding; Medac: Other: Travel support, Research Funding.

Background Recently, we demonstrated immunological and clinical responses to a RHAMM-R3 peptide vaccine in patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. To improve the outcome of the vaccine, a second cohort was vaccinated with a higher dose of 1,000 μg peptide.Design and Methods Nine patients received four vaccinations subcutaneously at a biweekly interval. Immunomonitoring of cytotoxic CD8+ as well as regulatory CD4+ T cells was performed by flow cytometry as well as by enzyme-linked immunospot (ELISpot) assays. Parameters of clinical response were assessed.Results In 4 of 9 patients (44%) we detected positive immunological responses. These patients showed an increase of CD8+RHAMM-R3_tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells and an increase of R3-specific CD8+ T cells. Two of these patients showed a significant decrease of regulatory T cells (Tregs). In one patient without response Tregs frequency increased from 5 to 16%. Three patients showed clinical effects: one patient with myelodysplastic syndrome RAEB-1 showed a reduction of leukemic blasts in the bone marrow, another myelodysplastic syndrome patient an improvement of peripheral blood counts and one patient with multiple myeloma a reduction of free light chains. Clinical and immunological reactions were lower in this cohort than in the 300 μg cohort.Conclusions High-dose RHAMM-R3 peptide vaccination induced immunological responses and positive clinical effects. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematologic malignancies. However, higher doses of peptide did not improve the frequency and intensity of immune responses in this trial. (This study is registered at http://ISRCTN.org as ISRCTN32763606)

Recent studies suggest inflammatory mechanisms involved in the pathogenesis of major psychiatric disorders (MPD). T cells play a major role during inflammation, but little is known about T cell subpopulations in the cerebrospinal fluid (CSF). We investigated the frequency of cells positive for the surface markers CD4, CD8, CD25, CD45, CD69, and CD127 in 45 paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples by multiparameter flow cytometry from patients with MPD of the schizophrenic and affective spectrum with normal CSF cell counts and compared them with those from patients with non-inflammatory (NIND), chronic inflammatory (CIND) neurological disorders, and meningitis (MEN). In MEN patients, CD4+ cell frequency in PB, but not in CSF, was significantly increased as compared to CIND and NIND. No difference between patient groups was observed for CD8+. CD4+CD45RO+ double positive cells in PB were significantly lower in CIND than in MEN or NIND. The frequency of CD4+CD25+ cells in PB was significantly higher in MEN than in MPD or CIND. For CSF, the percentage of CD4+CD127(dim) cells was significantly lower in MEN than in MPD. CD4+CD127(dim) in PB and CSF showed overlapping characteristic clusters between MPD and CIND and MEN patients. Overall, the hypothesis of low degree inflammation in a subgroup of MPD is supported. The analysis of lymphocyte subsets in PB and CSF constitutes a novel promising tool to understand underlying pathomechanisms in psychiatric and neurological disorders on an individual case level.

The aim of this retrospective study was to assess the safety and efficacy of bone marrow (BM) harvesting of allogeneic donors in an outpatient setting. Data of 226 related and unrelated donors who underwent BM harvest under general anesthesia at our institution from 2002 to 2014 were analyzed. Sixteen patients were a priori planned for admission for social reasons and 210 patients underwent BM harvesting with the intention to perform this procedure on an outpatient basis. To identify factors that predispose for hospital admission, we retrospectively analyzed donor characteristics and collection parameters. Outpatient treatment was performed in 178 of 210 donors (85%), whereas 32 donors (15%) required admission for clinical reasons (mainly clinically relevant anemia and circulatory problems). These individuals were not significantly different in sex distribution, age, donor's body weight, and the proportion of related donors from those who were not admitted. However, we found a significantly higher collection volume per kilogram donor's body weight in inpatients compared with volume for outpatients (16 versus 13 mL/kg body weight, P < .001). Severe adverse events or deaths occurred neither in the inpatient nor in the outpatient setting. Our study demonstrated that BM harvest in an outpatient setting is safe and feasible for the majority of allogeneic donors. A high volume of BM represented a major risk factor for inpatient admission.

Background and Objectives Biosimilars of the granulocyte colony stimulating factor (G-CSF) filgrastim were approved by the European Medicines Agency (EMA) for registered indications of the originator G-CSF, including prevention and treatment of neutropenia, as well as mobilization of peripheral blood stem cells in 2008. Nevertheless, there is still an ongoing debate regarding the quality, efficacy and safety of biosimilar G-CSF. Materials and Methods This article is a meta-analysis of clinical studies on the use of biosimilar G-CSF for mobilization and transplantation of haematopoietic stem cells as available in public databases. All data sets were weighted for the number of patients and parameters and then subjected to statistical meta-analysis employing the Mann–Whitney U-test followed by the Hodges–Lehmann estimator to assess differences between biosimilar and originator G-SCF. Results A total of 1892 individuals, mostly with haematological malignancies but also including 351 healthy donors have been successfully mobilized for autologous or allogeneic stem cell transplantation using biosimilar G-CSF (ZarzioTM: 1239 individuals; RatiograstimTM/TevagrastimTM: 653 individuals). A total of 740 patients with multiple myeloma, 491 with non-Hodgkin's lymphoma (NHL), 150 with Hodgkin's lymphoma (HL) and other diseases are included in this meta-analysis, as well as 161 siblings and 190 volunteer unrelated donors. For biosimilar and originator G-CSF, bioequivalence was observed for the yield of CD34+ stem cells as well as for the engraftment of the transplants. Conclusion Biosimilar G-CSF has equivalent effects and safety as originator G-CSF.

To give an overview over cell therapeutic approaches to immunosuppression in clinical kidney transplantation. A focus is on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). Literature review with an emphasis on already existing therapies. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness are now being tested in early phase I and phase II trials in clinical kidney transplantation. Cell products such as regulatory T cells or regulatory macrophages, or other myeloid suppressor cell therapies, may either consist of donor-specific, third-party, or autologous cell preparations. Major problems are the identification of the suppressive cell populations and their expansion to have sufficient amount of cells to achieve donor unresponsiveness (e.g., with regulatory T cells). We show a simple and safe way to establish donor unresponsiveness in living-donor kidney transplantation by MIC therapy. A phase I clinical trial is now under way to test the safety and efficacy of this cell therapeutic approach. Cell therapeutic approaches to immunosuppression after kidney transplantation may revolutionize clinical transplantation in the future.

The expression of tumor-associated antigens (TAAs) might play a critical role in the control of minimal residual disease (MRD) in acute myeloid leukemia (AML), and therefore might be associated with clinical outcome in AML. In a DNA microarray analysis of 116 AML samples, we found a significant correlation between high mRNA levels of G250/CA9 and longer overall survival (P = .022), a similar trend with high mRNA levels of PRAME (P = .103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1, we found no correlation with clinical outcome. High expression of at least 1 of the 3 TAAs, RHAMM/HMMR, PRAME, or G250/CA9, provided the strongest favorable prognostic effect (P = .005). Specific T-cell responses were detected in 8 (47%) of 17 patients with AML in complete remission for RHAMM/HMMR-R3 peptide, in 7 (70%) of 10 for PRAME-P3 peptide, and in 6 (60%) of 10 for newly characterized G250/CA9-G2 peptide, a significant increased immune response compared with patients with AML patients who had refractory disease (P < .001). Furthermore, we could demonstrate specific lysis of T2 cells presenting these epitope peptides. In conclusion, expression of the TAAs RHAMM/HMMR, PRAME, and G250/CA9 can induce strong antileukemic immune responses, possibly enabling MRD control. Thus, these TAAs represent interesting targets for polyvalent immunotherapeutic approaches in AML.

Background. The trifunctional bispecific antibody Removab (tbAB) bridges and activates CD3 positive T cells to EpCAM on carcinoma cells and simultaneously binds to an accessory immune-cell inducing tumor cell lysis. tbAB-induced tumor cytotoxicity was assessed in an autologous human ex vivo system. Methods. One hundred forty tumor samples and autologous peripheral blood mononuclear cells from a total of 36 patients with head and neck squamous cell carcinomas (HNSCCs) were incubated on a chicken embryo chorioallantois membrane with Removab. Tumor cells coincubated with cisplatin or cell culture medium served as positive and negative controls. Tumor cell lysis was assessed by acridine orange staining or by fluorescence-activated cell sorting of propidium iodide–marked cells after 24 and 48 hours (T24/T48) coincubation. Results. Coincubation of HNSCC cells with tbAB and autologous peripheral blood mononuclear cells resulted in a 49% ± 6% decrease of viable cells at T24 (p < .005) and in a decrease of 56% ± 8% at T48 (p < .005) compared with the control. The tumor cytotoxicity was similar to that of cisplatin (49% ± 7% decrease at T24 and 49% ± 8% at T48). Conclusion. In an autologous human ex vivo system, the tbAB-induced tumor cell lysis was comparable to that by cisplatin. © 2005 Wiley Periodicals, Inc. Head Neck27: XXX–XXX, 2005

Recently, the focus is on new specific immunotherapies for AML such as cellular therapies employing dendritic cells (DCs) generated from AML blasts. AML-DCs express constitutionally leukemia-associated antigens (LAAs) present in AML blasts they are generated from. Here we investigated whether the generation of AML-DCs would alter the expression level of LAAs. Moreover, we evaluated the presence of HLA and costimulatory molecules on AML blasts versus AML-DCs. Quantitative real-time polymerase chain reaction (PCR) was performed for the following LAAs: preferentially expressed antigen in melanoma (PRAME), the receptor for hyaluronic acid mediated motility (RHAMM/CD168), Wilms' tumor gene 1 (WT-1) and proteinase 3. The expression of HLA-ABC, HLA-DR, CD40, CD80, CD83 and CD86 was evaluated by flow cytometry. RHAMM protein expression was evaluated by immunocytochemistry, recognition of AML-DCs by PRAME epitope-specific T cells was evaluated in a chromium-release assay. Quantitative real-time PCR for AML-DCs versus AML blasts showed an alteration in mRNA expression of LAAs. An elevated PCR signal for PRAME was detected in 7/12 AML-DC preparations. 6/12 AML-DC preparations showed a significant upregulation of the PCR signal for RHAMM. A stronger WT-1 and proteinase-3 signal was observed in PCR for only 2/12 and 1/12 AML-DCs , respectively. All preparations showed a strong expression of at least one of the LAAs examined. As demonstrated by flow cytometry, AML-DCs strongly upregulated costimulatory molecules like CD40 and CD80 in comparison with AML blasts. AML-DCs tested positive for RHAMM protein. PRAME positive AML-DCs were recognized by specific T cells. AML-DCs might constitute a powerful tool in immunotherapy for AML. Real-time PCR allows a quick and quantitative assessment of immunologically relevant LAA expression with only 10(5) DCs and might be helpful for the decision whether the AML-DC vaccination strategy is favourable or not.

The present study investigated the effects of CD34(+) cell selection in 102 patients using the CliniMACS device. Patients were at high risk for the development of graft versus host disease (GvHD) because of age, or the use of a haploidentical, mismatched or unrelated donor (UD). The median age of the patients was 44 years. The CliniMACS procedure yielded 8.0 x 10(6) CD34(+) cells/kg and the number of residual T cells was 1.3 x 10(4)/kg (median). The median follow up was 20.6 months. The probability of graft failure was 7%. The rate of acute GvHD was low (compatible family donors 10%, UDs 17%, and haploidentical donors 26%) with no patient enduring more than grade II disease. The cumulative incidence of chronic GvHD at the median follow up after transplant was 15% for the compatible family donor group, 40% for the UD group and 78% in the group transplanted from a haploidentical donor Treatment failure was mainly because of transplant-related mortality, especially aspergillus infection, and not due to relapse. The probability of disease-free survival, stratified for the risk of treatment failure, was 27% for the high risk, 46% for the intermediate risk and 83% for the low risk group.