ALBERT

Description: Introduction: Patients receiving an allogeneic stem cell graft from a cytomegalovirus (CMV) seronegative donor are prone to CMV reactivation with high risk of disease and mortality. Therefore we initiated a clinical phase I trial with a novel vaccine designed by our group: a CMV phosphoprotein 65 (CMVpp65)-derived peptide in water-in-oil emulsion (Montanide™) plus administration of granulocyte-macrophage colony stimulating factor. Material and Methods: 10 patients after allogeneic stem cell transplantation received four vaccines s.c. at a biweekly interval. Patients were monitored for clinical course and CMVpp65 antigenemia. CMV-specific CD8+ and gamma/delta T cells were analyzed by multi-color flow cytometry. Neutralizing anti-CMV antibody assays were established and correlated to clinical parameters. Results: Peptide vaccination was well tolerated, no drug-related serious adverse events were detected. 7 of 9 patients with CMVpp65 antigenemia cleared the CMV with enduring response 〉 1.5 years after 4 vaccinations and are still free from antigenemia until present. 2 patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An up to 6-fold increase in frequency of both CMV-specific CD8+ T cells or Vdelta2- gamma/delta T cells with a maximum of 1.32% CMV-specific CD8+ T cells ex vivo without priming and 13.7% Vdelta2- gamma/delta T cells of all CD8+ T cells was detected in five patients. Also titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. Humoral and cellular immune responses correlated with clearance of the CMV load. Conclusion: Administration of CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing. Disclosures Kuball: Gadeta B.V.: Membership on an entity's Board of Directors or advisory committees. Wuchter:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy. Greiner:BMS: Research Funding.

Description: Key Points In AML with bialleleic CEBPA-mut relapse-free survival was improved by allogeneic and autologous hematopoietic stem cell transplantation. In relapsed patients second complete remission rate was high and survival was favorable after an allogeneic transplantation.

Description: Key Points Matching for MICA significantly reduces the incidence of acute and chronic GVHD in otherwise HLA 10/10-matched unrelated-donor HCT. Our results formally define MICA as a novel major histocompatibility complex-encoded human transplantation antigen.

Description: Transfer of tumor antigen–specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRα/β chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRα constant α (Cα) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Cα domain preceded by a TCRα signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Cα were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector ζ-chain–associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Cα transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Description: Grafting T cells by tumor-antigen specific T cell receptors (TCR) could trigger the initiation of effector function and redirect T cell cytotoxicity towards tumors. We utilized various HLA-A2.1 transgenic mice to bypass human MDM2- and p53-specific self-tolerance. In contrast to the use of HuCD8×A2Kb transgenic mice to generate an MDM2-specific CD8-dependent TCR, we generated a high-affinity, CD8-independent p53-specific TCR in single human A2.1 transgenic mice. The efficiency of double chain (dc) TCR modified T cells could be affected by the incorrect TCR α/β chain pairing between endogenous and transgenic TCR constructs to form hybrid TCR potentially leading to autoimmunity. To address this concern, chimeric A2.1-restricted peptide-specific murine single chain (sc) TCRs were constructed (Vα-Li-VβCβ) and retrovirally transduced into human T cells. Despite detectable surface expression, these chimeric receptors were not able to convey any MDM2- or p53-specific cytolytic activity. Therefore we developed a truncated TCR-alpha domain (Cα) comprising solely the TCRα signal peptide, the ecto-domain, the transmembrane region as well as the cytoplasmic tail and cotransduced these construct with the scTCRs. We anticipated that Cα would stabilize scTCR expression by interacting with the single chain beta chain. Indeed, this approach not only led to increased expression levels of the chimeric scTCRs, but also induced specific lysis of A2.1 positive MDM2 or p53 peptide-pulsed target cells as well as solid tumor cell lines. Recognition of malignant targets by p53 specific scTCR transduced CD4 and CD8-positive T cells was equivalent to that observed with double-chain p53 TCR gene modified effector cells. To test whether this concept is applicable to human TCRs as well, we constructed a human gp100-specific scTCR and a human Cα domain. In contrast to the gp100-specific double chain TCR, only a marginal expression pattern was observed for the human scTCR / Cα constructs. Introduction of an additional disulfide bond within the constant domains in order to stabilize TCR surface expression showed no effect. Since murine TCR are expressed on human T cells to a much higher extent, the human constant β-domain of the scTCR was replaced by murine Cβ. Comparable to the murine scTCR concept, the chimerized scTCR coexpressed with murine Cα demonstrated high cell surface expression and triggered cytotoxicity of malignant A2.1/gp100-positive targets. In summary, our results lay a commonly applicable conceptual basis for the construction of therapeutic scTCR to prevent recombination of natural and transgenic dcTCR alpha and beta chains.

Description: Major limitations of currently investigated αβT cells redirected against cancer by transfer of tumor-specific αβTCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous α or βTCR chains. Therefore, the ability of a defined γ9δ2TCR to redirect αβT cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a γ9δ2TCR efficiently reprograms both CD4+ and CD8+ αβT cells against a broad panel of cancer cells while ignoring normal cells, and substantially reduces but does not completely abrogate alloreactivity. γ9δ2TCR-transduced αβT cells reduced colony formation of progenitor cells of primary acute myeloid leukemia blasts and inhibited leukemia growth in a humanized mouse model. Thereby, metabolites of a dysregulated mevalonate pathway are targeted and the additional application of widely used biphosphonates is crucial for in vivo efficacy most likely because of its modulating effect on cytokine secretion of γ9δ2TCR-transduced αβT cells. Expression of NKG2D ligands and F1-ATPase contributed to the activity of γ9δ2TCR-transduced αβT cells but were not mandatory. In summary, γ9δ2 TCRs are an attractive alternative to broadly redirect αβT cells against cancer cells with both an improved efficacy and safety profile compared with currently used αβTCRs.

Description: CD137 is a member of the TNFR-family with costimulatory function. Here we show that it also has many favorable characteristics as a surrogate marker for antigen-specific activation of human CD8+ T cells. Although undetectable on unstimulated CD8+ T cells, it is uniformly up-regulated 24 hours after stimulation on virtually all responding cells regardless of differentiation stage or profile of cytokine secretion, which circumvents limitations of current surrogate markers for defining the repertoire of responding cells based on only individual functions. Antibody-labeled responding CD137+ cells can be easily and efficiently isolated by flow sorting or magnetic beads to substantially enrich antigen-specific T cells. To test this approach for epitope discovery, we examined in vitro priming of naive T cells from healthy donors to Wilms tumor antigen 1 (WT1), a protein overexpressed in various malignancies. Two overlapping pentadecamers were identified as immunogenic, and further analysis defined WT1(286–293) as the minimal amino acid sequence and HLA-Cw07 as the HLA restriction element. In conclusion, this approach appears to be an efficient and sensitive in vitro technique to rapidly identify and isolate antigen-specific CD8+ T cells present at low frequencies and displaying heterogeneous functional profiles, and does not require prior knowledge of the specific epitopes recognized or the HLA-restricting elements.

Description: Immunotherapy with innate immune cells has recently evoked broad interest as a novel treatment option for cancer patients. γ9δ2T cells in particular are emerging as an innate cell population with high frequency and strong antitumor reactivity, which makes them and their receptors promising candidates for immune interventions. However, clinical trials have so far reported only limited tumor control by adoptively transferred γ9δ2T cells. As a potential explanation for this lack of efficacy, we found unexpectedly high variability in tumor recognition within the physiologic human γ9δ2T-cell repertoire, which is substantially regulated by the CDR3 domains of individual γ9δ2TCRs. In the present study, we demonstrate that the reported molecular requirements of CDR3 domains to interact with target cells shape the physiologic γ9δ2T-cell repertoire and, most likely, limit the protective and therapeutic antitumor efficacy of γ9δ2T cells. Based on these findings, we propose combinatorial-γδTCR-chain exchange as an efficient method for designing high-affinity γ9δ2TCRs that mediate improved antitumor responses when expressed in αβT cells both in vitro and in vivo in a humanized mouse model.

Description: Key Points Clofarabine integrated in standard induction therapy for newly diagnosed AML reduces relapse probability but does not improve survival. Clofarabine improves survival in intermediate-risk AML categories ELN-1 and the AML genotype without NPM1 and without FLT3-ITD gene mutations.

Description: Abstract 2232 Poster Board II-209 Introduction Chronic graft-versus-host disease (cGVHD) is the major long term complication of allogeneic stem cell transplantation and shares similarities with auto-immune diseases, like systemic sclerosis (SSc).Although the contribution of donor T cells in the development of both acute and chronic GVHD is beyond doubt; the role of B cells is less defined. However, two observations suggest that B cells do contribute to chronic GVHD. First, autoantibodies can be detected in 15-90% of chronic GVHD patients. Furthermore, several small-scale retrospective clinical studies demonstrated that in vivo depletion of B cells with the CD20-specific Ab rituximab results in amelioration of chronic GVHD symptoms in heavily pretreated steroid-refractory patients. Objective In order to prospectively investigate B cell depletion as therapy for cGVHD, we have studied the efficacy of the CD20-specific antibody rituximab in patients with steroid-refractory extensive cGVHD. Methods In an ongoing prospective phase I/II study 15 patients with steroid-refractory extensive cGVHD were treated with rituximab (375 mg/m2 per week for 4 consecutive weeks). Clinical responses were monitored monthly for 1 year according to the NIH criteria (EudraCT 2008-004125-42). Results A total of 60 rituximab infusions were administered to 15 patients with steroid-refractory cGvHD. Toxicity was limited to one infectious event and one allergic reaction. Clinical responses were so far analyzed in 12 patients. Median follow-up was 6.5 months (range 1-13). Eight patients showed a partial response (67%) of which 5 patients (42%) had an ongoing response up to 1 year of follow-up and 3 patients (25%) relapsed. Two relapsed patients were retreated with rituximab 8 months after cessation of the initial rituximab treatment and showed again a partial response. In patients with deep sclerosis (n=10), sclerotic features disappeared completely or partially in 8 patients (80%). In patients with involvement of eyes (n=10), mucosa (n=4) and gastro-intestinal tract (n=4) response was observed in 50%, 25% and 25% of patients, respectively. Consequently, prednisone dosage could be reduced in 7 patients (58%). In 4 patients (33%) prednisone treatment was stopped completely. Conclusion Rituximab treatment is a feasible and effective treatment in patients with steroid refractory cGVHD. In addition, also patients with relapse of cGVHD after cessation of rituximab treatment can be successfully treated. Thereby, sclerotic lesions of the skin improved in the majority of patients whereas eye, mucosal and gastro-intestinal tract involvement was less reversible. We speculate that the lack of improvement of eye and mucosal involvement is a consequence of an early and irreversible destruction of lacrimal and salivary glands and this argues for an upfront application of rituximab in patients with cGVHD. Disclosures: Off Label Use: Rituximab, a monoclonal anti-CD20 antibody, is used to deplete B-cells in the treatment of chronic GVHD.