ALBERT

Description: T cell–mediated heterologous immunity to different pathogens is promising for the development of immunotherapeutic strategies. Aspergillus fumigatus and Candida albicans, the 2 most common fungal pathogens causing severe infections in immunocompromised patients, are controlled by CD4+ type 1 helper T (TH1) cells in humans and mice, making induction of fungus-specific CD4+ TH1 immunity an appealing strategy for antifungal therapy. We identified an immunogenic epitope of the A fumigatus cell wall glucanase Crf1 that can be presented by 3 common major histocompatibility complex class II alleles and that induces memory CD4+ TH1 cells with a diverse T-cell receptor repertoire that is cross-reactive to C albicans. In BALB/c mice, the Crf1 protein also elicits cross-protection against lethal infection with C albicans that is mediated by the same epitope as in humans. These data illustrate the existence of T cell–based cross-protection for the 2 distantly related clinically relevant fungal pathogens that may foster the development of immunotherapeutic strategies.

Description: Monoclonal antibodies have entered the therapy of multiple myeloma (MM) and are currently being evaluated in phase I-III trials. PAT-SM6 is a fully human IgM antibody that specifically binds to a cancer-specific cell surface variant of the chaperone molecule glucose regulated protein 78 (GRP78). Finding a GRP78 cancer-specific form on the surface of cancer cells, but not normal cells in vivo, presents an opportunity for cancer-specific targeting. This antibody is able to specifically target primary myeloma cells without showing cross-reactivity to healthy tissues (including plasma cells from healthy donors). Moreover, antibody treatment in vitro led to apoptosis in primary myeloma cells (Rasche L; PLOS One 2013). In vitro,PAT-SM6 was combined with Lenalidomide and/or Bortezomib and Dexamethasone in double and triple combinations on myeloma cell lines. Synergistic and additive cytotoxic effects were analyzed using the Chou-Talalay method. All double and triple combinations showed synergistic effect with a combination index (CI) 10mg/kg resulted in a significant reduction of plasma cells in the bone marrow (up to 54%) and a reduction of blood levels (up to 48%) of M protein. No cytotoxicity was observed. Based on these results we performed a Phase I clinical trial to examine the tolerability and safety of the PAT-SM6 antibody in patients with relapsed / refractory multiple myeloma. A pilot Phase I dose-escalating study was initiated (NCT01727778). Relapsed myeloma patients according to IMWG criteria were treated in different dose cohorts (0.3, 1,3 and 6mg/kg/dose) with at least four doses of PAT-SM6 as single agent in a two week cycle. A serological staging was performed on day 36. At the date of the abstract submission 9/12 subjects were treated. PAT-SM6 therapy was very well tolerated. No dose limiting toxicity (DLT), no related SAE and no related adverse events greater than grade 3 were observed. Mild leucopenia seemed to be a specific side effect. At date of submission 8 patients are evaluable for response. Two out of 8 patients showed stable disease according to IMWG criteria. In summary, PAT-SM6 provides a very promising approach for the immune therapy of patients with relapsed and refractory multiple myeloma. Disclosures: Braendlein: Patrys Ltd: Consultancy. Dubljevic:Patrys Ltd: Employment. Einsele:Celgene GmbH: Consultancy, Honoraria, Research Funding. Topp:Patrys Ltd: Honoraria.

Description: Abstract 2088 Introduction: A promising strategy for tumor therapy is the adoptive transfer of tumor specific T cells which are endowed with chimeric antigen receptors (CAR). First generation CARs are constructed by single chain antibodies and as signal domain the ζ chain of the CD3 complex. However, clinical trials are disappointing as adoptive transferred T-cells showed only modest persistence in patients resulting in limited clinical activity. We there for hypothesized that CAR expressing T-cells in comparison to unmodified T-cells display signaling defects when stimulated via their CARs. Methods: Cytomegalovirus(CMV)pp65 MHC I restricted CD8+ T-cells were generated, isolated by tetramer selection and modified with first generation CAR targeting CD19 and purified based on their receptor expression to more than 〉 95% purity. T-cell receptor (TCR) and CAR expression were quantified by Quantibright beads. Effector function of both T-cell populations were analyzed for specific lysis, cytokine production (IFN-g, TNF-a) and proliferation (CSFE) upon target cell stimulation. Phosphorylation of Erk, Jnk, p38 and PLC-γ was measured and analyzed with CBA Flexsets from BD. All statistical analyses have been performed using the statistical software package R. Signal peak intensities have been compared using the nonparametric wilcoxon rank sum test. Results: CMV-specific MHC-I restricted TCR as well as the CARs are expressed at same density levels and T-cells show equally lysis of targets either in the time of lysis onset as well the maximal lysis. In contrast, cytokine production (IFN, TNF-a) as well as antigen driven proliferation was reduced in CAR expressing T-cells when compared to CMV-specific CD8+ T-cells upon target exposure. PLC-γ was phosphorylated within minutes after target contact by CMV-specific CD8+ T-cells whereas CAR transduced CMV-specific CD8+ T-cells showed no significant phosphorylation of PLC-γ to target cell exposure. T-cell activated via CAR's demonstrated a statistically significant reduction of maximal phosphorylation in comparison to CMV-specific T-cells for ERK, for JNK and for p38. To exclude that CAR modification of CMV-specific CD8+ T-cells may impair signaling, CAR-CMV-specific CD8+ T-cells were exposed to CMVpp65 expressing targets. Killing, cytokine production and signal intensity were restored in comparison to parental CMV-specific CD8+ T-cells. Conclusion: CAR expressing T-cells show functionally signs of split anergy by efficient target elimination but fails to produce significant levels of cytokines and do not proliferate in response to target stimulation. Split anergy is not due to reduced expression of the CAR's but due to a complete lack of phosphorylation of PLC-γ as well as reduced phosphorylation of MAP-kinases ERK, p38 and JNK. These results potentially explain why primary CAR expressing T-cells fail to show significant clinical efficacy. Analysis of adequate phosphorylation, as proposed here, may be a powerful tool to identify the most promising second generation CARs for clinical studies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1945 Background Prognosis of patients with multiple myeloma (MM) has significantly improved by the introduction of autologous (auto) stem cell transplantation (SCT). The “novel drugs” which have shown activity in relapsed MM are increasingly used in first-line therapy aiming at maximized response prior to SCT. Whether allogeneic (allo) SCT adds to further disease control remains a matter of debate. Our group has shown the RAD regimen (lenalidomide, adriamycin and dexamethasone) to be highly effective and relatively well tolerated in relapsed and refractory MM. Therefore, we decided to explore RAD in the up-front management. Patients and Methods The current phase-II trial (DSMM XII) was designed to include patients (pts) up to the age of 65 years with newly diagnosed MM requiring treatment. We chose four cycles of RAD (lenalidomide 25 mg d-21; infusional adriamycin 9 mg/m2 per day d1-4; dexamethasone 40 mg d1-4 and 17–20; pegfilgrastim 6 mg d 6) every 4 weeks for induction followed by chemomobilization of peripheral blood stem cells. Low molecular weight heparin is mandatory during RAD treatment for thromboprophylaxis. All pts are to undergo one cycle of melphalan 200 mg/m2 followed by auto SCT. A subsequent allo SCT after reduced intensity conditioning (treosulfan/fludarabin) is scheduled for pts featuring at least one previously identified (cytogenetic or serologic) risk factor. Those with very favourable risk are to proceed to a second auto SCT. All patients will receive 12 months of lenalidomide maintenance (10 mg per day) on a continuous basis. Here, we present results of a planned safety analysis. Results 75 pts with a median age of 57 (range, 35–66) years have been enrolled by 11 German centers between 9/2009 and 7/2010. Currently, 51 pts are evaluable for toxicity during RAD induction: In all, 25 severe adverse events (SAEs) were reported for 16 subjects (31%). 68% of SAEs were assessed to be drug-related. Most frequent events were venous thrombosis (VTE; n=4), pyrexia (n=3) and syncope (n=2). Neutropenia, extravasation, pleural effusion, and allergic dermatitis accounted for one SAE each. 17 patients, 10 of whom (59%) had ISS stage II/III disease, are evaluable for post-induction response. Ten subjects (59%) achieved VGPR or better: 6 pts had VGPR and 2 patients each CR and stringent CR as assessed by the investigator. Conclusions Our preliminary results suggest RAD to be a well tolerated and effective novel induction protocol in up-front treatment of MM. Notably, incidence of severe hematotoxicity observed so far is significantly lower than was in our previous study in relapsed/refractory pts. Incidence of VTE was acceptable while no neurotoxicity occurred. Updated results will be presented. Disclosures: Knop: Celgene Germany: Consultancy, Honoraria. Off Label Use: Lenalidomide in combination with doxorubicin in myeloma first-line therapy. Reichle:Celgene Germany: Research Funding. Einsele:Celgene Germany: Consultancy, Honoraria. Bargou:Celgene Germany: Consultancy, Research Funding.

Description: Introduction: The standard treatment for patients with relapsed or refractory (r/r) classical Hodgkin Lymphoma (cHL) is salvage chemotherapy followed by autologous stem cell transplantation (ASCT). The best outcome from the transplant is expected in patients who achieve complete remission (CR) after the salvage chemotherapy. Everolimus is a mammalian target of rapamycin (mTOR) inhibitor that has shown activity and an acceptable toxicity profile in patients with r/r cHL (Johnston PB et al. Am J Hematol 2010; Johnston PB et al. ISHL-9 2013). We therefore aim to improve the CR rate after salvage by an enforced salvage regimen consisting of everolimus plus time-intensified standard DHAP (Dexamethasone, High-Dose AraC [Cytarabine], Cisplatinum). Methods: We conducted a phase I trial of everolimus plus DHAP (ever-DHAP) for patients with r/r cHL eligible for ASCT to assess the recommended phase II dose (RPTD) of oral everolimus in this combination. Everolimus was administered for 14 days in each DHAP cycle starting from one day before DHAP. The second cycle of ever-DHAP was scheduled to start at day 14 of cycle one. Patients received two cycles of ever-DHAP. Everolimus dose levels of 2.5mg, 5mg, 7.5mg and 10mg were assessed in a modified 3+3 design. Generally, dose-limiting toxicity (DLT) was defined as CTCAE grade III/IV toxicity or unsuccessful stem cell mobilization. However, grade III/IV non-hematological toxicities known from DHAP were only counted as DLT from second occurrence in any patient onwards. Grade III/IV hematological toxicities were only counted as DLTs in case of prolonged time to recovery. Restaging by (PET)-CT was performed at day 21 of the second cycle given that the patient had recovered; PET was mandatory for all patients not achieving a CR in the CT scan. The rate of patients experiencing DLTs during 2 cycles of the combination therapy was the primary endpoint of the study. Secondary endpoints included adverse events, tumor related results of therapy or death, treatment administration, time to recovery after end of treatment and stem cell mobilization. Results: 14 patients were recruited between August 2012 and January 2014, all patients received at least one dose of everolimus. The median age was 33.5 years; six were female and eight were male. Eleven patients presented with stage III/IV disease. Overall, treatment was well tolerated. Two patients (both at the 7.5mg level) had a premature treatment termination and did not receive the 2nd cycle, one patient due to ototoxicity of grade I at investigator’s discretion and one patient due to neurotoxicity of grade III/IV. One additional patient (7.5mg cohort) suffered from ototoxicity of grade III/IV between end of treatment and restaging. Both grade III/IV toxicities were pre-defined as known from DHAP, thus they were not considered dose limiting at this first occurrence. No further non-hematological grade III/IV adverse events were reported. All patients but one experienced grade III/IV thrombocytopenia and leukopenia. No DLTs occurred and therefore 10mg of everolimus/day was chosen as RPTD. Duration of induction therapy including restaging after two cycles was 35-54 days. All patients who completed the 1st treatment cycle (n=13) had adequate stem cell mobilization (CD34+ cell count 2.9 - 31.1 x 106/kg; median 10 x 106/kg). So far one death occurred in a patient with pneumococcal sepsis one year after restaging. Responses according to CT scan in 12 patients who received two cycles of ever-DHAP were CR in 3 patients, CR unconfirmed (CRu) in 3 patients, partial remission (PR) in 5 patients and stable disease (SD) in 1 patient. This accounts for a CR/CRu rate of 50% (6/12) and an overall response rate of 92% (11/12). A PET scan was performed in 4 of 6 patients with CR/CRu; all had a negative PET (Deauville 1). In the 6 patients not achieving a CR/CRu 5 patients had a PET scan; 4 were PET positive (2 patients with Deauville 4 and 5 each) and 1 was PET negative (Deauville 1). Conclusions: Ever-DHAP with 10mg everolimus/day is safe and feasible in patients with r/r cHL. Based on the results of this phase I trial a randomized, placebo-controlled trial of ever-DHAP has been initiated and is currently recruiting. Disclosures von Tresckow: Takeda: Honoraria, Travel grants, Travel grants Other; Novartis: Honoraria, Research Funding. Off Label Use: Everolimus in relapsed or refractory Hodgkin Lymphoma. Topp:Affimed: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria. Engert:Millennium: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Affimed: Consultancy. Borchmann:Takeda: Honoraria, Research Funding, Travel grants Other.

Description: Introduction: AFM13 is a bispecific, tetravalent TandAb antibody that targets CD16A and CD30. Preclinical data demonstrated that AFM13 specifically harnessed NK cells to exert potent cytotoxic effects on tumor cells expressing CD30. Furthermore, AFM13 showed a favorable toxicity profile in cynomolgus monkeys. HL is characterized by the presence of CD30+ Reed-Sternberg cells and by a high medical need in the r/r setting, especially since the response to brentuximab vedotin in this patient group is mostly short. Here, we present the final results of a phase 1 clinical study in r/r HL including a focus on the pre-treatment with brentuximab vedotin. Methods: Heavily pretreated patients (pts.) with r/r HL received stepwise escalated doses of intravenous AFM13 (0.01 to 7.0 mg/kg) weekly or 4.5 mg/kg twice weekly over 4 weeks. Primary objectives were safety and tolerability, secondary objectives included pharmacokinetics (PK), pharmacodynamics (PD), and clinical efficacy measured using Cheson criteria. Results: 28 pts. were recruited, 24 pts. received AFM13 weekly and 4 pts. received AFM13 twice weekly. AFM13 was well tolerated with mainly mild to moderate adverse events. The maximum tolerated dose was not reached. AFM13 demonstrated activity, which was consistently more pronounced for all assessed parameters at doses ≥1.5 mg/kg (n=13). 3 of 13 pts. (23%) exhibited partial responses and disease control was reached in 10 of 13 pats. (77%). Importantly, AFM13 was also active in pts. refractory to brentuximab vedotin as most recent treatment. PK data revealed an AFM13 half-life of up to 19 hours. In peripheral blood, the portion of activated NK cells (CD69+), relative to total number of NK cells, increased up to 3-fold 24 hours after infusion compared to baseline. Kinetics of NK cell activation corresponded to kinetics of AFM13 serum levels. Soluble CD30 declined to zero at the end of treatment in almost all pts. Conclusion: Phase 1 data indicate that AFM13 is active and well tolerated in heavily pretreated r/r HL pts. including brentuximab vedotin failures. To further enhance efficacy of AFM13 both, the dose regimen (based on PK/PD profile) and the duration of treatment, will be optimized in a phase 2 study. This study is currently in preparation by the German Hodgkin Study Group. Disclosures Topp: Affimed Therapeutics AG: Consultancy. von Tresckow:Takeda: Honoraria, Other; Novartis: Honoraria, Research Funding. Marschner:Affimed Therapeutics AG: Employment. Engert:Millenium Takeda: Consultancy; Affimed Therapeutics AG: Consultancy.

Description: Introduction: Relapsed/refractory (r/r) B-precursor ALL in adults has an unfavorable prognosis with a median overall survival of 4–8 months and a 5-year survival of

Description: Introduction Regulatory T cells (Tregs) are checkpoint cells for the success or failure of an adaptive immune response in malignant diseases. Currently the influence of Tregs enumeration on the outcome of T cell mediated immune therapy has so far not been assessed in the clinical context of novel bispecific T cell engaging antibody construct blinatumomab. Blinatumomab as single agent is able to induce a hematologic remission of 46.7% in patients with relapsed and refractory B-ALL across two completed trials with a total of 225 patients (Topp et al. ASH 2012, ASCO 2014). We therefore addressed the question if quantification of Tregs numbers alone, as well as in combination with other factors can predict the outcome of blinatumomab therapy in r/r ALL patients. Furthermore, we determine the mechanism how Tregs might influence the immune response. Material and Methods T cell compartment was immune monitored by multiclor FACS analyses in 31 patients treated in both completed blinatumomab r/r ALL trials on day 0 prior to blinatumomab treatment. A decision tree approach was applied to obtain a first overview of the covariate structure in relation to the classification of the responder and non-responder group. A logistic regression model was fitted including all significant covariates after a preselection based on the statistical significance of a wilcoxon rank sum test. Model covariates have further been selected by a step-down approach starting with the full model. For the evaluation of the influence of blinatumomab on Tregs, activation markers were measured by FACS staining. For quantification of cytokines of the blinatumomab engaged Tregs, CBA technology was used. Proliferation und suppression assays were performed with CFSE dilution technique. Treg depletion was performed with MACS bead separation. Results At our center 16 out of 31 r/r ALL (51.6%) patients reached a CR/CRh* after two cycles of blinatumomab. Patients who were refractory to the treatment had a median of 16.1% (8.4-73%) Tregs, whereas responders had median of 8.55% Tregs (3.8-14.2%) (p value: 0.00013). LDH was significantly increased (median 773 U/l) prior to treatment in non-responders in comparison to the responder group (median 206 U/l) (p-value of 0.00532). Other parameters like age, bone marrow blast cells, number of CD3, CD3/Tregs ratio, previous allogeneic hematopoietic stem cell transplantation and gender were also tested. Multivariate logistic regression analysis included percentage of Tregs and LDH as the most significant covariates. Based on cross-validation, the model yielded an estimated prediction performance of 83.8% for the correct classification of patients benefiting from the therapy (responders). Analogously, the primary split in the tree based classification analysis was defined by the Tregs predicting non-responders on a level of 12.15% or higher with an internal accuracy of 92% (11/1). The second split turned out to be LDH, which further sub-classified responders with LDH 〈 324.5 with 100% accuracy. In order to decipher why high amounts of Tregs had an adverse effect on the success of blinatumomab, in vitro studies with Tregs were performed. Tregs incubated with blinatumomab and primary ALL blasts upregulated CD69, CD25 and PD1 and produced 270pg/µl of IL-10 but only traces of Th1 cytokines when compared to CD4/CD25- Th cells. Tregs cocultured with blinatumomab coated primary ALL blasts could also suppress in a dose dependent manner the proliferation of autologous CD4CD25- T cells with a maximal suppression of 50%. In three peripheral blood samples of blinatumomab refractory r/r ALL patient with high Tregs percentage, Tregs were depleted by CD39 MACS depletion. In all three Treg depleted patient samples, after adding ALL cells and blinatumomab, proliferation was significantly restored when compared to samples where Tregs were not depleted. Conclusion The percentage of regulatory T cells in combination with LDH prior to blinatumomab therapy predicts in 83.8% as a biomarker the response of blinatumomab in r/r ALL patients. Nevertheless, the predictive value of Treg numbers has to be confirmed in a prospective trial. The underlying mechanism involves activation of Tregs by blinatumomab coated ALL blasts resulting in secretion of IL-10 and suppression of proliferation. Proliferation of T-cells can be restored by upfront removal of Tregs and may be a strategy to convert r/r ALL blinatumomab non-responder to responder. Disclosures Einsele: Celgene GmbH: Consultancy, Research Funding. Topp:Amgen: Consultancy, Honoraria, Other.

Description: Abstract 2326 In patients undergoing hematopoietic stem cell transplantation (HSCT) infectious complications are frequent causing substantial morbidity and mortality. Adoptive T cell therapy specific for single pathogens has previously shown to efficiently control viral and fungal infections but approaches targeting multiple pathogens are limited to T cells generated with EBV transformed B cells that are genetically modified expressing multiple viral antigens. Infections are often experienced by different viral and fungal pathogens such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (AdV), Aspergillus fumigatus (AF) and Candida albicans (CA) that show a wide spectrum of memory T cell frequencies. Those with a low precursor frequency are not suitable for selection methods based on the secretion of cytokines such as IFN-g. As CMV seropositivity among HSCT donors may range only between 30–40% and immunity to the other pathogens can be detected simultaneously in more than 85% of HSCT donors we focused on the generation of a multi-specific T cell product for EBV, AdV, AF and CA for easy transfer under current regulatory requirements. We aimed to develop a simple protocol which (i) is able to enrich T cells specific for pathogens with low precursor frequency and (ii) allows simultaneous expansion of multiple pathogen-specific T cells in a single culture. We determined if CD154, which is transiently expressed on antigen stimulated CD4+ but also to a lesser extend on CD8+ T cells, would be a potential candidate for selection of pathogen-specific T cells. For stimulation we used peptide pools for AdV hexon protein, EBV latent membrane protein 2 (LMP2) and CA mannose protein 65 (MP65) as well as one AF immune dominant epitope derived from the Crf1 protein. To select and expand antigen-specific T cells, we stimulated PBMC for 16 hours, separated them by CD154+ MicroBead Kit (Miltenyi) and co-cultured them with irradiated autologous PBMC with IL-2, IL-7 and IL-15 for 14 days. The isolated cells were on average 0.62% of the starting fraction and could be expanded 20- to 145-fold. The median frequency of AdV-specific T cells increased from day 1 to day 14 87-fold from 30 to 2620 spot forming counts (SFC)/2×105 cells, for EBV 229-fold from 15 to 3430 SFC/2×105 cells and for CA 960-fold from 3 to 2400 SFC/2×105 cells assessed by IFN-γ ELISPOT. AF-specific T cells that were undetectable in PBMC increased to a median of 2260 SFC/2×105 cells. Although isolation of CD154+ cells favors enrichment of CD4+ T cells, a low fraction of virus-specific CD8+ T cells were simultaneously expanded. Next, we tested the efficacy of the CD154-based enrichment for the generation of multi pathogen-specific T cell lines reactive to all 4 pathogens. Selection and expansion was comparable, there was however a notable shift in the frequencies of T cells specific for different antigens in multi pathogen-specific cultures compared to single lines. The median increase of AdV-and CA- specific T cell lines was comparable (2345 SFC/2×105 and 3205 SFC/2×105 cells) but the frequencies for EBV (575 SFC/2×105 cells) as well as for AF (465 SFC/2×105 cells) were diminished in multi-specific lines. Nevertheless, lysis of LCL pulsed with LMP2 or AdV peptide pools was efficient with 72% and 36% by single and 30% and 45% by multi-specific T cell lines (at an E:T ratio of 20:1) as assessed by 51Cr-release assay. The single and multi pathogen-specific T cell lines generated by peptides responded to endogenously processed antigens and were able to specifically proliferate upon antigen stimulation. In contrast, T cell-mediated allo-reactivity was almost abrogated when compared to the starting population. In conclusion, we established a simple expansion protocol for selection, expansion and enrichment of allo-depleted single and multiple pathogen-specific CD4+ and CD8+ T cells specific for AdV, EBV, AF and CA that may further expand if the T cells are stimulated by their native antigen in vivo. This expansion protocol may form the basis for adoptive immunotherapy trials in HSCT recipients at risk for multiple infectious complications. This study has been supported by a grant of BayImmunet. Disclosures: No relevant conflicts of interest to declare.