ALBERT

Description: Abstract 4173 Introduction: Chronic graft-versus-host disease (cGVHD) following allogeneic hematopoietic stem cell transplantation remains the most important cause of late non-relapse mortality. While first-line treatment of cGVHD can be based on controlled data evidence for second-line treatment remains challenging with no single agent having been approved for salvage treatment yet. Here, we report a dual center retrospective cross-sectional analysis on salvage treatment of refractory cGVHD with everolimus, an inhibitor of the mammalian target of rapamycin (mTOR). Patients and Methods: A total of 78 consecutive patients (51 males, 27 females) with a median age of 53 years (range: 20 – 71 years) who received everolimus for off-label treatment of refractory cGVHD were considered eligible for this analysis. Potential toxicities according to Common Terminology Criteria for Adverse Events (CTCAE) were assessed in a retrospective manner using patients' records. Response to treatment was assessed using the cGVHD Global Severity Score according to NIH Consensus Criteria (NIH Severity Score). Possible response rates included complete remission (CR), partial remission (PR), mixed response (MR), stable disease (SD) and progressive disease (PD). Results: Out of 78 total patients 14 (18%) suffered from mild, 38 (49%) from moderate, and 26 (33%) from severe cGVHD before being commenced on an everolimus-based salvage regimen. Concomitant immunosupression included steroids, calcineurin inhibitors, inosine monophosphate dehydrogenase inhibitors, methotrexate and/or rituximab. Patients received daily oral doses of everolimus ranging from 0.125 to 6 mg resulting in median plasma levels of 4.1 ng/mL (range: 1.1 – 7.4 ng/mL). At time of analysis median follow-up after introduction of everolimus was 731 days (range: 14 – 2205 days). Median treatment duration with everolimus was 484 days ranging from 12 to 1250 days. Everolimus-based treatment was discontinued in 49 patients due to toxicity (n=14), CR (n=12), PR (n=6), MR (n=6), death (n=6), refractory cGVHD (n=3), relapse of underlying disease (n=1) or patient's request (n=1) while 29 patients (37%) were continued on everolimus. Most frequent grade 3/4 toxicities included infections (n=29) and thrombocytopenia (n=15). Infectious complications were the main cause of death. There was a single case of relapse of the underlying malignancy in the entire treatment series. NIH Severity Score improved in 34 patients (44%), remained stable in 36 patients (46%), and worsened in 8 patients (10%) resulting in a 21% CR and 31% PR rate of cGVHD. The total sum of NIH Severity Scores in all patients assessable was significantly reduced after treatment with everolimus (168 vs. 125; p

Description: Abstract 4056 Allogeneic hematopoetic stem cell transplantation (aHSCT) is a treatment option for a variety of diseases in particular heamatological malignancies. Due to an ongoing immunosuppression to prevent graft versus host reaction (GvHD), disease relapses as well as viral infections are major causes of morbidity and mortality after aHSCT. T cell responses against different tumor-associated and tumor-specific antigens could be detected not only in patients with malignant diseases but also in healthy donors. We investigated the selection of MART-1 (Melanoma Antigen Recognized by T-cells), Proteinase 3 and WT-1 (Wilms Tumor- Antigen) specific T cells from the blood of healthy donors as basis for a tumor-specific T cell transfer in the context of aHSCT. With a newly established protocol, based on streptamer selection, we isolated simultaneously multi-functional and multi-specific T-cell populations. We selected tumor-antigen-specific CTL′s (Cytotoxic T- Lymphocytes) mentioned above and also antiviral T-cells namely against CMV, EBV and AdV from a single blood donation. In this simultaneous selection with up to 7 different epitopes in one step, even the tumor specific T cells, which are known to be rarely detected among healthy donors, could be enriched to an amount sufficient for a direct T cell transfer. Purity achieved after selection was at least 82% and up to 98,87%, which minimizes the risk for GvHD after clinical application. The possibility to transfer these selected CTL`s to patients after stem cell transplantation improves the graft versus tumor effect as well as the anti infective T cell immunity without a relevant risk for GvHD. Furthermore, the selected multi specific T cell populations could be expanded in vitro without loss of specificity and include different phenotypes such as central memory and memory effector cells, which may provide long lasting immunity. Moreover, starting with a leukapheresis, we successfully transferred our selection protocol in a closed system according to current good manufacturing practice (cGMP) requirements, which allows clinical application in the future. With that, it opens new perspectives in cellular immunotherapy against malignancies and viral infection for patients after aHSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4351 Wilms‘ tumor protein-1 (WT1) is one of the most investigated tumor-associated antigens (TAA) in hematological malignancies. CD8 T-cell responses against several WT1-derived peptides have been characterized and are known to contribute to disease control after allogeneic hematopoietic stem cell transplantation (HSCT). Also the identification of human leukocyte antigen (HLA) class II-restricted CD4 T-cell epitopes from WT1 is a challenging task of T-cell-based cancer immunotherapy to improve the effectiveness of WT1 peptide vaccination. We found a highly immunogenic WT1 peptide composed of only 9 amino acids having the ability to induce IFN-γ secretion in CD4 T-cells in an HLA DR-restricted manner. This finding is of great interest as it was generally accepted that HLA class II binding peptides are composed of at least 12 amino acids being recognized by CD4 T-cells, whereas HLA class I binding peptides are composed of 8–11 amino acids being recognized by CD8 T-cells (Wang et al Mol. Immunol. 2002). However, both HLA class I and class II molecules bind to primary and secondary peptide anchor motifs covering the central 9–10 amino acids. Thus, considering this common structural basis for peptide binding there is a possibility that the WT1 9-mer peptide binds to HLA class II molecules, and induces CD4 T-cell responses. IFN-γ induction in response to several WT1 9-mer peptides was screened in 24 HLA-A*02:01 positive patients with acute myeloid leukemia or myelodysplastic syndrome after allogeneic HSCT. Responses to one WT1 9-mer peptide were exclusively detected in CD3+CD4+ T-cells of 2 patients after allogeneic HSCT, but not in CD3+CD4+ T-cells of their corresponding HSC donors. CD4+ T-cell responses to this WT1 9-mer peptide exhibited high levels of functional avidity, as IFN-γ induction was detected after stimulation with 100 ng peptide per mL. Peptide-induced IFN-γ production was confirmed with IFN-γ ELISPOT assays and the HLA restriction of the T-cell response was determined by HLA blocking antibodies. The reaction was significantly blocked by anti-pan HLA class II antibody (85 % reduction), but neither by pan-HLA class I nor by anti-HLA A2 antibody. To identify the subtype of HLA class II molecule, blocking assays with antibodies against HLA-DP, HLA-DR and HLA-DQ were performed. IFN-γ induction was completely abrogated by anti-HLA-DR antibody (99 % reduction) (fig 1, p value of unpaired student‘s t-test

Description: Abstract 4650 Introduction MVA is one of the most promising vaccine candidates for infectious diseases as well as for malignancies. Astonishingly, little information exists about the mechanism by which immune responses to MVA are generated. It was shown that among leukocytes - which are essential for the generation of cellular and humoral immune responses - APCs like dendritic cells, monocytes and B cells are preferentially infected. But little is known about the effects on APCs. Therefore we wanted to analyze in more detail the general effects of MVA infections on different immune cells. Methods Primary human peripheral blood mononuclear cells (PBMCs) and isolated leukocyte populations in particular monocyte derived DCs, monocytes and B cells were infected with (wildtype) wtMVA or MVA-gfp (green fluorescent protein) to verify the infection. Subsequently changes in surface markers and cytokine expression were assessed. Results Among PBMCs and specific isolated leukocyte populations, monocytes, DCs and B cells were most susceptible to MVA infection. NK cells showed a lower and T cells a very low infection rate. Surprisingly, selected monocytes were less susceptible to MVA as compared to unselected ones. This phenomenon is due to phagocytosis of other infected PBMCs by monocytes. Furthermore we could show that MVA causes a downregulation of CD14 on iDCs and monocytes as well as of CD25, CD80, and CD86 on B cells. Furthermore, there was a slight downregulation of CD1a on iDCs and mDCs and of CD80 on iDCs. On the other hand MVA caused an upregulation of HLA-DR on monocytes and additionally a slight upregulation of CD40 on iDCs. Moreover, MVA evoked a slight upregulation of CD83 on iDCs but a slight downregulation on mDCs. Above all, we could demonstrate that MVA induces an upregulation of CXCL10 in iDCs, mDCs, monocytes, and B cells, and an upregulation of TNFα, IL-6, and IL-12p70 in iDCs, mDCs, and monocytes. In addition, we revealed a downregulation of CXCL8 in monocytes as well as of IL-β in B cells. Conclusions These results suggest that MVA induces a Th1-polarized immune response in APCs. Thus, MVA seems to be an appropriate vaccine vector for antiviral immunotherapy of stem cell transplant recipients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4737 Flow cytometry has become a routine method in both clinical and basic immunological research. Its ability to differentiate between distinct populations of cells by surface staining of various parameters is a main advantage since we have the possibility to identify antigen-specific T-cells by flow cytometry through the development of soluble multimeric peptide–MHC complexes. Nevertheless, surface staining does not provide information about the functionality of the analyzed cell populations. Hence, further methods have been described to define cells by detection of intracellular epitopes. These assays include the intracellular staining of distinct cytokines or phosporylated signaling molecules (Phosflow). MHC-multimer approaches combined with intracellular cytokine staining are routinely used, whereas the detection of intracellular p-kinases under MHC-multimer staining applying the Phosflow-protocols has not been realized so far. The use of phosphoepitope analysis in antigen-specific T-cells is of high interest in infections or especially during immunosuppressive drug treatment. Therefore, we aimed to establish a dual multimer-phospho-staining protocol to provide a method to get insight into the biochemical signaling processes in antigen-specific T-cells. We chose CTL responses against CMV as model system due to well established epitopes and high frequency in healthy donors. The original Phosflow-protocols did not turn out to be suitable for a combination with MHC-multimer staining. The very harsh fixation and permeabilization procedures largely or completely abrogated the antigen-specific staining. We have been able to stain both the CMV-specific T-cell-receptor and phosphorylated kinases following polyclonal stimuli (e.g. PMA, IL-2 etc.) using different protocols for some p-kinases (ERK, STAT5, NfKB, p38). These protocols allow a combination of specific T-cell-receptor staining with that of intranuclear phosphoepitopes after polyclonal stimulation. In preliminary experiments, we have also been able to show a specific phosphorylation of the ERK molecule after stimulation with CMV-specific artificial antigen-presenting cells or antibody-coated plates. As mentioned above, the use of phosphoepitope analysis in antigen-specific T-cells may offer the possibility to correlate immunological anergy with distinct signaling processes in defined clinical situations, e.g. in immunosuppressed patients post alloSCT. Disclosures: No relevant conflicts of interest to declare.