ALBERT

Description: Identification of tumor-associated antigens (TAA) has resulted in the development of therapeutic vaccines for the treatment of cancer. We applied an integrated functional genomics approach to identify TAA in malignant tissues of patients with renal cell carcinoma (RCC). A comparative DNA chip analysis of tumor and the corresponding non-malignant tissue from patients with RCC followed by sequencing of peptides bound to the HLA-class I molecules by mass spectrometry was applied to identify novel TAA in RCC. To confirm the immunogenicity of identified epitopes cytotoxic T lymphocytes (CTL) were generated using dendritic cells (DC). Utilizing this approach, two peptides derived from RGS5 binding to either HLA-A*02 or 03 were identified. The key function of regulators of G protein-signalling (RGS) is to bind to G protein α subunits and to stimulate their intrinsic GTPase activity. The hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) thereby is accelerated and the inactive heterotrimer more rapidly restored. Thus, RGS proteins inhibit the biological activity of G proteins. Interestingly, it was recently shown that RGS5 is overexpressed in pericytes of newly developing tumor vessels, indicating that RGS5 plays an important role during tumor angiogenesis. Using RT-PCR analysis we found that RGS5 is expressed on a broad variety of tumor cells including RCC, colorectal, breast and ovarian cancer, malignant melanoma and multiple myeloma as well as in acute and chronic leukemias making this protein an interesting candidate for the development of vaccination strategies to target the tumor cells and the tumor vessels. CTL that were induced using the RGS5 peptides lysed autologous DC pulsed with the cognate peptide or transfected with in vitro transcribed RGS5 RNA as well as HLA-matched tumor cell lines. The specificity and HLA restriction was confirmed using blocking monoclonal antibodies and in cold-target inhibition assays. We next utilized DC transfected with RGS5 RNA to generate specific CTL. Using this approach we confirmed the processing and presentation of the identified peptides by malignant cells. These CTL lysed tumor cells in an antigen specific manner while sparing non-malignant cells. To analyze the induction of RGS5 specific CTL in an autologous setting in patients with malignant diseases, we used blood samples from a patient with acute myeloid leukemia (AML) in complete remission after chemotherapy and were able to generate specific CTL capable of recognizing autologous AML blasts while sparing non-malignant cells. Our results demonstrate that RGS5 is a novel tumor rejection antigen expressed in a wide range of malignancies that can be applied to target malignant cells and tumor angiogenesis.

Description: Abstract 4096 Poster Board III-1031 We previously demonstrated the immunogenicity of a combined vaccine approach employing two leukemia-associated antigenic peptides, PR1 and WT1 (Rezvani Blood 2008). Eight patients with myeloid malignancies received one subcutaneous 0.3 mg and 0.5 mg dose each of PR1 and WT1 vaccines in Montanide adjuvant, with 100 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF). CD8+ T-cell responses against PR1 or WT1 were detected in all patients as early as 1 week post-vaccination. However, responses were only sustained for 3-4 weeks. The emergence of PR1 or WT1-specific CD8+ T-cells was associated with a significant but transient reduction in minimal residual disease (MRD) as assessed by WT1 expression, suggesting a vaccine-induced anti-leukemia response. Conversely, loss of response was associated with reappearance of WT1 transcripts. We hypothesized that maintenance of sustained or at least repetitive responses may require frequent boost injections. We therefore initiated a phase 2 study of repeated vaccination with PR1 and WT1 peptides in patients with myeloid malignancies. Five patients with acute myeloid leukemia (AML) and 2 patients with myelodysplastic syndrome (MDS) were recruited to receive 6 injections at 2 week intervals of PR1 and WT1 in Montanide adjuvant, with GM-CSF as previously described. Six of 7 patients completed 6 courses of vaccination and follow-up as per protocol, to monitor toxicity and immunological responses. Responses to PR1 or WT1 vaccine were detected in all patients after only 1 dose of vaccine. However, additional boosting did not further increase the frequency of PR1 or WT1-specific CD8+ T-cell response. In 4/6 patients the vaccine-induced T-cell response was lost after the fourth dose and in all patients after the sixth dose of vaccine. To determine the functional avidity of the vaccine-induced CD8+ T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of PR1 and WT1 peptides (0.1 and 10 μM) was measured by IC-IFN-γ staining. Vaccination led to preferential expansion of low avidity PR1 and WT1 specific CD8+ T-cell responses. Three patients (patients 4, 6 and 7) returned 3 months following the 6th dose of PR1 and WT1 peptide injections to receive a booster vaccine. Prior to vaccination we could not detect the presence of PR1 and WT1 specific CD8+ T-cells by direct ex-vivo tetramer and IC-IFN-γ assay or with 1-week cultured IFN-γ ELISPOT assay, suggesting that vaccination with PR1 and WT1 peptides in Montanide adjuvant does not induce memory CD8+ T-cell responses. This observation is in keeping with recent work in a murine model where the injection of minimal MHC class I binding peptides derived from self-antigens mixed with IFA adjuvant resulted in a transient effector CD8+ T cell response with subsequent deletion of these T cells and failure to induce CD8+ T cell memory (Bijker J Immunol 2007). This observation can be partly explained by the slow release of vaccine peptides from the IFA depot without systemic danger signals, leading to presentation of antigen in non-inflammatory lymph nodes by non-professional antigen presenting cells (APCs). An alternative explanation for the transient vaccine-induced immune response may be the lack of CD4+ T cell help. In summary these data support the immunogenicity of PR1 and WT1 peptide vaccines. However new approaches will be needed to induce long-term memory responses against leukemia antigens. To avoid tolerance induction we plan to eliminate Montanide adjuvant and use GM-CSF alone. Supported by observations that the in vivo survival of CD8+ T-effector cells against viral antigens are improved by CD4+ helper cells, we are currently attempting to induce long-lasting CD8+ T-cell responses to antigen by inducing CD8+ and CD4+ T-cell responses against class I and II epitopes of WT1 and PR1. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 515 We established a highly efficient GMP-grade, ex-vivo selective allodepletion process where host-activated donor T cells are eliminated based on their preferential retention of the photosensitizer 4,5-dibromorhodamine 123 (TH9402) and exposure to visible light (Kiadis Pharma, The Netherlands). As relapse of disease largely impairs the overall success of allogeneic stem cell transplantation we aimed to improve this outcome by using selectively T cell depleted allografts in order to reduce post transplant immunosuppression and thereby enhance graft versus malignancy effects. To determine the appropriate level of post transplant immunosuppression we designed a three sequential de-escalation stage trial with grade III-IV acute GvHD as the primary endpoint involving 17 patients per study cohort. Here we report on the first completed study cohort of NIH trial 07-H-0136 where seventeen patients (median age 44 (28-68) years) with hematological malignancies received a CD34-selected (Miltenyi, Germany) stem cell allograft together with 5 × 106/kg selectively depleted donor T cells following an age-adapted, radiation-based preparative regimen (FluCyTBI). Eleven patients had high risk disease (including ALL (Ph+, CR〉1), refractory NHL, AML/MDS, AML with chloroma and blast crisis CML). Low-dose cyclosporine was used as sole immunosuppression for 90 days post transplant in the absence of GvHD. At a median follow-up of 385 (119-714) days actuarial probabilities (±SEM) of acute GvHD were 35±12% for grade II-IV and 0% for grade III-IV. Non-relapse mortality (NRM) was low with 17±11%. Overall survival (OS) was 73±12% and relapse-free survival (RFS) was 65±13% with a relapse probability of 21±11% (Figure). A low relapse incidence in a high-risk population suggests functionality of selectively allodepleted T cells. The absence of severe GvHD reflects the efficacy of the allodepletion process. Based on these findings we have initiated recruitment for the next study cohort where post transplant immunosuppression will be limited to 45 days only. Ultimately the aim is to achieve further reduction in immunosuppression in the absence of severe GvHD in order to enhance graft versus malignancy effects and thereby improve the outcome after allogeneic stem cell transplantation especially for patients at high risk for relapse. Disclosures: Mielke: Kiadis Pharma, The Netherlands: Research Funding, The current trial is supported under a clinical trial agreement between NHLBI and Kiadis.. Savani:Kiadis Pharma Inc., The Netherlands: Consultancy. Barrett:Kiadis Pharma, The Netherlands: The current trial is supported under a clinical trial agreement between NHLBI and Kiadis..

Description: Background: AMD3100 (AMD or plerixafor) inhibits the binding of SDF-1 to its cognate receptor CXCR4 resulting in the rapid mobilization of CD34+ hematopoietic progenitor cells into the peripheral circulation. A prior phase I study suggested peak CD34+ cell mobilization occurred 6–8 hours following a 240 mcg/kg dose of AMD. This investigation provides preliminary data on the safety and CD34+ cell mobilizing effects of doses up to 480 mcg/kg of AMD. Methods: To account for inter-subject variability, subjects in three cohorts received two different subcutaneous doses of AMD. Healthy volunteers received the following doses of AMD: 240 mcg/kg and 320 mcg/kg (cohort 1); 320 mcg/kg and 400 mcg/kg (cohort 2); and 400 mcg/kg and 480 mcg/kg (cohort 3). Subjects received the higher dose at least 14 days after the first to allow adequate wash-out of AMD. The absolute number of circulating CD34+ cells were measured after each dose at the following times: 0, 2, 4, 6, 8, 10, 12, 14, 18, and 24 hours. In addition, this study assessed the following parameters: adverse events, AMD pharmacokinetics (400 and 480 mcg/kg doses), cytokine polarization status (Th1 vs Th2) of AMD mobilized CD3+ T-cells, and endothelial progenitor cell measurements. Results: To date, 16 of the planned 18 subjects have been enrolled on study with final results available for the first two cohorts; preliminary results exist for four subjects in the third cohort (400 mcg/kg vs 480 mcg/kg dose). Common toxicities include diarrhea, nausea, sinus tachycardia, injection site redness, perioral paresthesias, and headache. No dose limiting toxicities were observed; however, the frequency and/or magnitude of headache, nausea, vomiting, diarrhea, and tachycardia were greater at the 400 mcg/kg dose. Table 1 summarizes the preliminary CD34+ mobilization results for the 240, 320, and 400 mcg/kg doses. The lines in figures 1 and 2 show the difference in an individual subjects peak CD34+ cell number mobilized following 2 different doses of AMD. Conclusion: Although this phase I study is ongoing, preliminary data suggest doses of AMD higher than 240 mcg/kg may improve the peak numbers of CD34+ cells mobilized into the circulation. Furthermore, although dose escalation continues, the adverse events observed after a 400 mcg/kg dose of AMD appear similar to those observed after lower doses; no grade 3 or 4 adverse events have been observed. The final study results will provide additional data on the dose of AMD that optimizes CD34+ cell and CFU-GM mobilization for both autologous and allogeneic hematopoietic stem cell collections. CD 34+ cell mobilization 240 mcg/kg 320 mcg/kg 400 mcg/kg *Two subjects in 400 mcg dose group had the same peak mobilization time at two time points: these were averaged Number of subjects 6 12 10 Mean mobilized CD34 cells/microliter 27 29 31 Standard deviation of CD34 cells/microliter 13 14 13 Average peak mobilization time in hours* 9 10 11 Range of peak mobilization times in hours 8−14 6−14 8−18 Figure Figure

Description: We describe the safety and immunogenicity of a combined vaccine of 2 leukemia-associated antigenic peptides, PR1 and WT1. Eight patients with myeloid malignancies received one subcutaneous dose each of PR1 and WT1 vaccines in Montanide adjuvant, with granulocyte-macrophage colony-stimulating factor. Patients were reviewed weekly for 4 weeks to monitor toxicity and immunologic responses. Toxicity was limited to grades 1 to 2. Using peptide/HLA-A*0201 tetramers and intracellular interferon-γ staining, CD8+ T cells against PR1 or WT1 were detected in 8 of 8 patients after a single vaccination. To monitor the kinetics of vaccine-induced CD8+ T-cell responses and disease regression after vaccination, absolute PR1 and WT1+CD8+ T-cell numbers and WT1 expression were studied weekly after vaccination. Responses occurred as early as 1 week after vaccination. After vaccination, the emergence of PR1 or WT1+CD8+ T cells was associated with a decrease in WT1 mRNA expression as a marker of minimal residual disease, suggesting a vaccine-driven antileukemia effect. Conversely, loss of response was associated with reappearance of WT1 transcripts (P 〈 .01). This is the first demonstration that a combined PR1 and WT1 vaccine is immunogenic. These results support further studies of combination immunization strategies in leukemia patients. This study is registered at http://clinicaltrials.gov as NCT00313638.

Description: Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from allografts to prevent graft versus host disease (GvHD) while conserving useful donor immunity. We developed a semi-closed, GMP-quality, clinical scale SD process where donor-derived lymphocytes are stimulated with patient-derived T-cell antigen presenting cells in an ex vivo mixed lymphocyte reaction (MLR). Alloactivated donor T cells preferentially retain the photosensitizer 4,5-dibromorhodamine 123 (TH9402), rendering them susceptible to elimination by exposure to visible light in a photodepletion device (Kiadis Pharma Inc, The Netherlands). After Food and Drug Administration and Institutional Review Board approval we initiated a clinical trial where HLA-identical sibling recipients with hematological (non T-cell) malignancies received a CD34-cell selected transplant (Miltenyi, Germany) containing less then 1 × 104 T cells/kg together with 5 × 106/kg viable SD donor T cells on day 0, using an age-adapted, radiation-based preparative regimen (FluCyTBI). Low-dose cyclosporine was used as sole immunosuppression in the absence of GvHD. Eleven patients (median age 43 (28–68) years with ALL, MDS, CML, mantle cell lymphoma (MCL), or AML) were transplanted with a median follow-up of 240 (43–400) days. Nine patients were considered high risk. Patients received a stem cell product containing a median of 6.0 (3.9–9.5) ×106/kg CD34+ stem cells in addition to 5×106/kg SD T cells. Absolute lymphocyte recovery was rapid (median 834 (384–2486) cells/μL day 30 post transplant) [Fig A]. Early T cell chimerism was donor-dominated (median 66% (6–95) on day 14, and 97% (82–100) on day 30, and 100% (92–100) on day 45 [Fig B]. One patient received an unmanipulated DLI to treat a delayed fall in T cell chimerism. Three patients developed steroid-sensitive grade II aGvHD of skin (N=2) and gut (N=1) but no grade III–IV aGvHD occurred after transfusion of the photodepleted lymphocytes [Fig C]. Two patients developed limited chronic GvHD. Only one patient, transplanted for refractory MCL, relapsed 340 days after transplant. One patient died of infectious complications and GvHD 330 days after transplant after receiving an unmanipulated DLI in her home country for suspected, but subsequently unconfirmed relapse. Eight patients reactivated CMV but were successfully treated. These results demonstrate for the first time clinical feasibility of photodepletion-based SD stem cell allotransplants in matched siblings. Robust lymphocyte recovery and early donor chimerism with a low relapse incidence in a high-risk population suggest functionality of SD T cells in the absence of severe GvHD, which should allow further reduction of immunosuppression to optimize disease control in future studies.

Description: Abstract 1017 Poster Board I-39 On patent and off-patent drugs with previously unrecognized anti-cancer activity, could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we compiled and screened a chemical library for potential anti-leukemia agents. From these screens, we identified the antihelmintic flubendazole that is currently used for the treatment of gastrointestinal and systemic parasites, but has not been evaluated for the treatment of malignancy. To explore its efficacy as an anti-cancer agent, leukemia and myeloma cell lines were treated with increasing concentrations of flubendazole. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Flubendazole reduced cell viability with an LD50 ≤ 1 μM in 8/8 myeloma and 4/6 leukemia cell lines, a concentration that is pharmacologically achievable. Likewise, flubendazole reduced the clonogenic growth of primary AML samples at nanomolar concentrations. Cell death was confirmed by Trypan blue staining. Given the effects in leukemia and myeloma cells lines, we evaluated the effects of flubendazole in mouse models of leukemia and myeloma. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 leukemia or OPM2 myeloma cells. Mice were then treated intraperitoneally with flubendazole (20-50 mg/kg/day – doses more than 10-fold lower than the LD50) or buffer alone. Flubendazole decreased tumor weight and volume in both mouse models up to 5-fold compared to control without evidence of weight loss or gross organ toxicity. Mechanistically, flubendazole inhibited bovine-tubulin polymerization in cell-free assays and disrupted microtubule architecture in intact cells as visualized by confocal microscopy. We demonstrated that flubendazole bound tubulin at the colchicine binding site, a region distinct from where vinca-alkaloids bind. Flubendazole arrested cells in the G2 phase of the cell cycle and increased the number of multi-nucleated cells. We also demonstrated that cell death after flubendazole treatment was related to its ability to inhibit microtubule polymerization by using cell lines with tubulin mutations (gifts from Dr F. Loganzo, Wyeth, Pearl River, NY and Drs. S. Band Horwitz and C. Yang, Albert Einstein College of Medicine, Bronx, NY). Vinca-alkaloids are p-glycoprotein (Pgp) substrates and Pgp over-expression can limit the efficacy of these agents. Therefore, we tested the effects of Pgp over-expression on flubendazole's cytotoxicty. CEM-VBL cells over-expressing Pgp remained fully sensitive to flubendazole, but were over 1000-fold more resistant to vinblastine than wild type CEM cells. Therefore, flubendazole can overcome some forms of vinca-alkaloid resistance. Given that flubendazole binds tubulin at a site distinct from vinca-alkaloids, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. OCI-AML2 leukemia cells were treated with increasing concentrations of flubendazole and vinblastine. Flubendazole and vinblastine synergistically induced cell death with combination index (CI) values of 0.09, 0.017, 0.003 and 0.001 at the EC 50, 25, 10 and 5, respectively, where CI values

Description: Abstract 4748 Introduction Radiofrequency (RF) ablation is a safe and effective method to locally treat malignancies of the liver, kidney and more recently lung. For the treatment of small tumors, long term results are comparable with outcome after surgical resection and better than current chemotherapies. RF-ablation has now been reported to induce heat shock protein (HSP) expression on sublethally damaged hepatocytes. HSPs are potent alarm signals for the immune system causing i.e. activation of T cells and dendritic cells. In addition to triggering immune responses directed against HSPs themselves, HSPs can carry antigens derived from those cells of which HSPs originated and thereby elicit specific immune reactions against these cellular epitopes. Patients and Methods We here analyzed serum samples of 22 patients to evaluate the systemic release of HSP70 using a specific HSP70 ELISA. Patients were followed for a median of 30 months until death or three years after the last RF-treatment. As a control group, sera from 20 patients undergoing diagnostic liver biopsy were screened. Results The median increase of HSP70 in serum comparing levels before and one day after the procedures was 1.6-fold in the RF-ablation group, while no soluble HSP-70 could be detected in the control group before and after the biopsy. Nine patients showed a higher than two-fold increase of HSP70 in the serum after ten sessions of RF-ablation compared to levels prior to treatment. No correlation of HSP70 levels with tumor volume, the ablated non-malignant tissue or the total size of the necrosis was observed. In the subgroup of patients with elevated HSP-70 serum levels, 67% (6 out of 9) were alive at the end of follow up with only one suffering from progressive disease. In contrast, 61% (8 out of 13) of the other patients suffered from progressive disease or had deceased at the end of follow-up. Conclusion RF-ablation not only induces cellular HSP expression but also can lead to a systemic release of serum HSP70. Patients with strong increases in HSP-70 serum levels tended to have a better survival than patients without induction of HSP-70 in the serum. Thus, immunomodulation might represent an additional beneficial effect of RF-ablation besides local tumor destruction, since HSPs are strong stimuli for the immune system and can mediate tumor-specific T cell immunity. Disclosures: No relevant conflicts of interest to declare.