Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3 of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3 end rather than one extended with unrelated nucleotides, and 2 sequential ␤-globin 3 untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4 ؉ and CD8 ؉ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations. IntroductionAntigen-encoding RNA 1,2 has the advantages of a genetic vaccine (delivery of all epitopes of the whole antigen, easy manufacturing, standardized purification) and the added value of a safe pharmaceutical characterized by transient expression and lack of integration into the genome of the treated host. The combination of this versatile antigen delivery molecule with dendritic cells (DCs) as the most potent antigen-presenting cells is regarded as an attractive approach to induce cellular and potentially therapeutic immune responses in patients with cancer. Reports demonstrated convincingly that the use of RNA results in efficient induction of antigen-specific immune responses in vitro and in animal models 1,[3][4][5][6][7] and paved the way for trials in humans. Early clinical trials showed feasibility, lack of toxicity, and promising efficacy based on immunologic and clinical read-outs. [8][9][10][11][12] At this early phase of clinical development, antigen-specific RNA has the status of a drug compound requiring detailed exploration.Basic pharmacologic issues that must be addressed in drug development include the pharmacokinetics of the compound of interest within the system of its physiological activity after administration. In the quoted clinical trials, this system would be represented by immature or mature DCs. A key objective of such investigations is better understanding of the impact of the structural features of the drug formulation on its pharmacologic properties. Neither of these questions has thus far been addressed for antigen-e...

Hematopoietic stem cell transplantation (HSCT) from haploidentical donors is a viable option for patients lacking HLA-matched donors. Here we report the results of a prospective multicenter phase I/II trial of transplantation of TCRαβ and CD19-depleted peripheral blood stem cells from haploidentical family donors after a reduced-intensity conditioning with fludarabine, thiotepa, and melphalan. Thirty pediatric and 30 adult patients with acute leukemia (n = 43), myelodysplastic or myeloproliferative syndrome (n = 6), multiple myeloma (n = 1), solid tumors (n = 6), and non-malignant disorders (n = 4) were enrolled. TCR αβ/CD19-depleted grafts prepared decentrally at six manufacturing sites contained a median of 12.1 × 106 CD34+ cells/kg and 14.2 × 103 TCRαβ+ T-cells/kg. None of the patients developed grade lll/IV acute graft-versus-host disease (GVHD) and only six patients (10%) had grade II acute GVHD. With a median follow-up of 733 days 36/60 patients are alive. The cumulative incidence of non-relapse mortality at day 100, 1 and 2 years after HSCT was 5%, 15%, and 17% for all patients, respectively. Estimated probabilities of overall and disease-free survival at 2 years were 63% and 50%, respectively. Based on these promising results in a high-risk patient cohort, haploidentical HSCT using TCRαβ/CD19-depleted grafts represents a viable treatment option.

We report the first prospective, multi-center, open-label, single-arm phase I/II clinical trial that assesses the safety and feasibility of stem cell transplantation with TCRalpha/beta and CD19-depleted haploidentical grafts generated with the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a reduced-intensity conditioning in pediatric patients suffering from various malignant and non-malignant diseases (www.clinicaltrialsregister.org; 2011-005562-38). All patients received single agent MMF as short-term GVHD prophylaxis (40mg/kg/day for 30 days). The speed of immune reconstitution was measured in two core labs using standardized methods and the MACSQuant flow cytometry device (Miltenyi Biotec, Germany). Results: Thirty patients from six hospitals were treated (13 female, 17 male; median age 7 years, range 1 - 17 years). Of the 30 recipients, 10 had ALL, 8 had AML, 6 had solid tumors (soft tissue sarcomas and neuroblastomas), 3 had MDS/MPS, and 1 each with lysosomal storage disorder, SCID, and Wiskott Aldrich syndrome. Disease status in acute leukemias/MDS was: CR1 (n=4), relapsed/refractory (n=17). 5/6 patients with solid tumors had relapsed metastatic disease. The conditioning regimen consisted of 15 or 30 mg ATG (Fresenius/Grafalon) or 7 Gy total nodal irradiation, 160 mg/m2 fludarabine, 10 mg/kg thiotepa, and 140 mg/m2 melphalan. The median number of CD34+ cells, TCRalpha/beta+ cells and CD20+ cells infused was 14.6 x 106 (range, 4 - 54.9), 14 x 103 (range, 0.62 - 40.6) and 0.55 x 105 (range, 0.04 - 1.85), respectively. In addition, significant numbers of NK and TCRgd+ cells/kg were infused - 6.67 x 107 (median; range, 0.68 - 18.2) and 1.58 x 107 (median; range, 0.13 - 4.7), respectively. All 30 patients tolerated the infusion of haploidentical stem cell grafts well. Twenty-five patients had primary engraftment of ANC > 500 cells/µL at a median of 12 days (range, 10 - 18) and PLT > 20,000 cells/µL at a median of 15 days (range, 11 - 27). Peripheral T-cell chimerism at the time of engraftment was completely donor in 19/25 patients (76%), mixed in 3 (12%), and not measured in three. Five patients experienced primary graft failure and 2 had secondary graft failure. All except of one were successfully re-transplanted. None of the recipients developed severe acute GVHD grades III - IV. Only 1 patient had acute GVHD grade II that started on day 22. The vast majority of patients (96.7%) experienced no or only grade I acute GVHD despite minimal GVHD prophylaxis after transplantation. Samples from 24/25 patients with primary engraftment were evaluable for immune reconstitution (Figure 1). On day 28, the majority of WBC were NK cells (median 309 cells/µL; range, 64 - 1026). The second main type of cells were CD3+ cells (median 151 cells/µL; range, 9 - 953), mostly TCRgd+ (median 87 cells/µL; range, 7 - 891). At day 100, TCRab+ cells equalled TCgd+ cells (median 108 vs. 116 cells/µL). B cells recovered more slowly, with a median of 255.5 cells/µL (range, 1 - 1218) on day 63. ADV reactivation contributed most to infectious complications following transplantation. In total, 16/30 patients had ADV DNAemia or were positive in stool. Additionally, seven patients were tested positive for CMV (blood or urine). BK virus was present in 5 patients with 3 patients experiencing cystitis. No EBV reactivation was observed. Two patients had bacterial sepsis, 1 moderate, 1 fatal (due to non-engraftment).No fatal viral infection occurred within 100 days. One molecular relapse was observed within 100 days post transplantation that was treated with blinatumomab. Two of the 30 transplanted patients died within 100 days after transplantation: 1 patient due to sepsis following graft failure (non-relapse mortality) and 1 due to relapse. On day 100, chimerism was completely donor in 20 patients and mixed in two. Conclusions: The CliniMACS depletion system of TCRab+ and CD19+ cells yielded a large number of CD34+ cells, NK cells and TCRgd+ cells, that could be infused safely into pediatric patients with minimal risk of severe acute GVHD. The immune reconstitution was rapid and there was no TRM associated with viral or fungal infections. Coupled with a reduced-intensity regimen, the overall TRM was low. Longer follow up will provide essential information on chronic GVHD and survival outcomes. Figure 1 Immune Reconstitution after transplantation of TCR-alpha/beta and CD19 depleted haploidentical stem cell grafts Figure 1. Immune Reconstitution after transplantation of TCR-alpha/beta and CD19 depleted haploidentical stem cell grafts Disclosures Bader: Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Medac: Consultancy, Research Funding. Karitzky:Miltenyi Biotec: Employment. Holtkamp:Miltenyi Biotec: Employment. Siewert:Miltenyi Biotec: Employment. Bönig:Miltenyi Biotec: Consultancy, Honoraria, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-Patentholder of TcRalpha/beta depletion technology.

Background CD19 redirected chimeric antigen receptor (CAR) T-cell therapy has proven efficacy in relapsed or chemotherapy-refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL). However, targeting a single B-cell antigen leads to selective pressure with potential antigen-escape and subsequent relapse. A tandem CAR targeting CD20 and CD19 (pLTG1497) has been developed to overcome this limitation. Preclinical evaluation showed improved anti-lymphoma activity. Thus, we initiated a first-in-human, phase I clinical study of autologous pLTG1497-transduced CAR T-cells (MB-CART2019.1) in r/r B-NHL patients. Aims In this phase I prospective multi-center trial (NCT03870945) we aimed to evaluate the maximum tolerated dose (MTD) of MB-CART2019.1 in adult patients with CD20 and CD19 positive r/r B-NHL as determined by dose limiting toxicities (DLTs). Methods This was a 6+3 trial design with two predefined dose levels (DL1 1x106 and DL2 2.5x106 CAR T-cells/kg body weight, respectively). Secondary endpoints included adverse events (AEs) and best overall response rate (ORR). Pharmacodynamic assessments included maximum concentration (Cmax) of CAR T-cells, time to peak expansion (tmax), AUC (d0 to d28), and persistence. MB-CART2019.1 was produced by lentiviral transduction of autologous fresh leukapheresis in the closed automated CliniMACS Prodigy® System (Miltenyi Biotec, Bergisch Gladbach, Germany). Re-infusion (Day 0) of fresh MB-CART2019.1 was scheduled 14 days after leukapheresis. Fludarabine/cyclophosphamide lymphodepleting chemotherapy was administered from day -5 to -3. Results A total of 12 patients, 6 per dose level have been enrolled and treated between February and December 2019, 5 female and 7 male patients. Median age was 72 y (range 20, 78 y), with 10 patients >65 y and 8 >70 y. Histologies included aggressive B-NHL (11) and mantle cell lymphoma (1). Five (5) patients had refractory disease at study entry and IPI was ≥3 in 7 patients. Median time from leukapheresis to re-infusion was 14 d (range 13, 14 d). No DLT and no cytokine release syndrome (CRS) or neurotoxicity grade ≥3 were observed. One patient in dose level 1 experienced a grade 5 AE, which was due to disease progression. CRS grade 1 occurred in 3/6 patients on DL1 and DL2 each, and CRS grade 2 in 2 patients on DL2. Tocilizumab was given in 1 patient. Neurotoxicity grade 1 occurred in 1 patient on DL2. The above described CRS and neurotoxicity resolved completely. Mean Cmax of MB-CART2019.1 was 348.3 cells/µl (range 3.9, 830.4 cells/µl) on DL1 and 692 cells/µl (range 5.3, 3147.8 cells/µl) on DL2. Mean tmax was 15.8 d (range 9, 21 d) on DL1 and 11.5 d (range 9, 14 d) on DL2. Mean AUC was 3155 d*cells/µl (DL1) and 4339 d*cells/µl (DL2). Persistence of MB-CART2019.1 was observed in 12/12 patients until data cut-off. Altogether 9/12 patients (ORR 75%) responded to MB-CART2019.1 with 5/12 CRs. In DL1 3/6 patients responded (ORR 50%) and in DL2 6/6 patients (ORR 100%). The 3 patients without response to MB-CART2019.1 had a mean AUC0-28 of 870 d*cells/µl, whereas mean AUC0-28 in 9 responders was 4843 d*cells/µl reflecting the correlation between the pharmacodynamic parameters and the clinical response. Responses are ongoing in 5/9 patients, with a maximum duration of response of 330 days at data cut-off. Summary/Conclusions In this first-in-human dose finding study of MB-CART2019.1 no DLT and no severe (grade ≥3) CRS or neurotoxicity were observed. Feasibility and safety were very good in this cohort of elderly r/r B-NHL patients. The sustained expansion of tandem CAR T-cells was accompanied by efficacy: all patients (6/6) treated on DL2 responded and all 5 patients with CR (5/5) are in ongoing remission by the time of this report. Based on the promising risk-to-benefit ratio observed in our study, evaluation of MB-CART2019.1 at a dose of 2.5x106/kg body weight in clinical phase II and phase III trials for patients with relapsed aggressive B-NHL is underway. Disclosures Borchmann: Miltenyi Biotec B.V. & Co. KG: Honoraria. Balke-Want:Miltenyi Biotec B.V. & Co. KG: Honoraria. Ayuk:Celgene: Consultancy, Honoraria; Kite/Gilead: Honoraria; Therakos/Mallinckrodt: Honoraria, Research Funding; Neovii: Research Funding; Novartis: Honoraria. Holtkamp:Miltenyi Biomedicine GmbH: Current Employment. Preussner:Miltenyi Biomedicine GmbH: Ended employment in the past 24 months. Zadoyan:Miltenyi Biomedicine GmbH: Current Employment. Hanssens:Miltenyi Biomedicine GmbH: Current Employment. Kaiser:Miltenyi Biotec B.V. & Co. KG: Current Employment. Jurk:Miltenyi Biotec B.V. & Co. KG: Current Employment. Bürger:Miltenyi Biotec B.V. & Co. KG: Current Employment. Schneider:Lentigen Technology Inc., A Miltenyi Company: Current Employment, Patents & Royalties. Dropulic:Lentigen Technology Inc., A Miltenyi Company: Current Employment. Overstijns:Miltenyi Biomedicine GmbH: Current Employment, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec B.V. & Co. KG: Current Employment, Membership on an entity's Board of Directors or advisory committees. Scheid:Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria; Takeda: Honoraria, Research Funding. Holtick:Miltenyi Biotec B.V. & Co. KG: Honoraria. Miltenyi:Miltenyi Biomedicine GmbH: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Lentigen Technology Inc., A Miltenyi Company: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Miltenyi Biotec B.V. & Co. KG: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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