ALBERT

Description: Abstract 578 Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to explore the role of immunotherapeutic strategies in eradicating residual disease. A fundamental challenge to developing an effective anti-tumor immune response is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Key elements contributing to tumor-mediated immune suppression are the increased presence of regulatory T cells in patients with malignancy, and upregulation of the PD-1/PDL1 pathway. Tumor expression of PD-L1 promotes T cell tolerance by binding PD-1 on activated T cells and suppressing their capacity to secrete stimulatory cytokines. In addition, the PD-1/PDL-1 pathway has been shown to inhibit T cell-mediated lysis of tumor cells, potentially preventing a clinically meaningful immunologic response to tumor vaccines. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (CT-011) alone (Cohort 1) and in combination with a dendritic cell/myeloma fusion cell vaccine (Cohort 2) following ASCT. To date, 27 patients have been enrolled into Cohort 1, in which patients receive three infusions of CT-011 at doses of 3mg/kg given at 6 week intervals beginning 1–3 months following ASCT. Mean age of the patients is 57 years; 61% are male. 11 patients have received at least two infusions of CT-011. The remaining patients are undergoing pre-transplant therapy/transplant. CT-011 has been well tolerated, with possibly related adverse events consisting of transient grade 1–2 leukopenia, diarrhea, fatigue, arthralgia, rash, and peri-orbital edema. One patient developed grade 3 neutropenia, which resolved after two days without growth factor. Immunologic response was determined by quantifying circulating tumor reactive T cells prior to each dose of CT-011 and at 1, 3, 6 months following the last infusion, as defined by the percentage of T cells expressing IFNg in response to ex vivo exposure to autologous tumor lysate. 4 patients have completed 6 months of follow up after the third dose of CT-011, and are evaluable for immune response. CT-011 therapy was associated with the dramatic expansion of myeloma specific T cells. Mean percentage of circulating tumor reactive CD4+ and CD8+ T cells increased from 1.5 and 1.96 respectively prior to the first infusion of CT-011, to 4.26 and 8.28 respectively 1 month following the third infusion. As determined by tetramer staining in the subset of patients who are HLA A2.1, infusion of CT-011 resulted in a mean 9 fold expansion of T cells specific to the MUC1 antigen, which is aberrantly expressed by myeloma cells. Notably, immunologic response to CT-011 persists at 6 months following completion of therapy. Clinical response, as determined by time to disease progression, will be determined with longer follow up, as the median time from transplant is presently 8 months. We are initiating enrollment to Cohort 2, in which patients will be vaccinated with an autologous DC/myeloma fusion vaccine 1 week prior to each dose of CT-011. These data demonstrate that CT-011 results in the expansion of tumor reactive lymphocytes in the early post-transplant period, providing an ideal platform for combination with a tumor vaccine. Disclosures: Rosenblatt: CureTech Ltd.: Research Funding. Schickler:CurTech Ltd.: Employment, Research Funding. Rotem-Yehudar:CureTech Ltd: Employment, Research Funding. Avigan:CureTech Ltd: Research Funding.

Description: Introduction Our group has pioneered a personalized vaccine in which patient-derived acute myeloid leukemia (AML) cells are fused with autologous dendritic cells (DC/AML fusion), presenting a broad array of leukemia associated antigens with DC mediated costimulation. In a clinical trial of AML patients who were vaccinated after chemotherapy-induced remission, 71% remained free of disease at median follow up of 57 months. We sought to identify factors associated with durable remission after vaccination using genomic analysis of the bone marrow microenvironment including single cell RNA-seq and TCR clonal diversity analysis. Methods Banked bone marrow samples both prior to and 1 month post-vaccination were selected from patients who maintained long disease remission for greater than 5 years and those who had early relapse. FFPE marrow core biopsy samples (N=10) were the source for gene expression analysis. NEBNext ultra II directional library prep kit and Illumina NextSeq 500/550 system were used to generate reliable high quality RNA sequencing data. Differentially expressed genes were identified by p-value (≤0.01) and fold change (≥2) using Linear Models for Microarray (Limma) approach. Ingenuity Pathways IPA 9.0 was then used to define pathways and upstream regulators. Flash frozen samples (N=4) were analyzed by RNAseq at the single cell level using a standard 10X genomics approach with cell cluster annotation performed with Single Cell Wizard software. Banked peripheral blood was used to evaluate TCR diversity with Takara SMART-Seq next-generation sequencing to amplify variable regions of TCR- α/β subunits. Results Heatmaps depict significant differential gene expression in bone marrow biopsies both pre- and post-vaccination in patients who remained in long-term remission (responders) compared to those who relapsed (non-responders). Prior to vaccination there was modest upregulation of immune activation pathways including IL-7, IL-17A as well as inhibition of TGF-b in responders, suggesting a role of the micro-environment in modulating response. Significantly upregulated pathways in responders after vaccination (p value

Description: Fas belongs to the family of type-1 membrane proteins that transduce apoptotic signals. In the present studies, we characterized signaling during Fas-induced apoptosis in RPMI-8226 and IM-9 multiple myeloma (MM) derived cell lines as well as patient plasma cell leukemia cells. Treatment with anti-Fas (7C11) monoclonal antibody (MoAb) induced apoptosis, evidenced by internucleosomal DNA fragmentation and propidium iodide staining, and was associated with increased expression of c-jun early response gene. We also show that anti-Fas MoAb treatment is associated with activation of stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK); however, no detectable increase in extracellular signal-regulated kinases (ERK1 and ERK2) activity was observed. Because interleukin-6 (IL-6) is a growth factor for MM cells and inhibits apoptosis induced by dexamethasone and serum starvation, we examined whether IL-6 affects anti-Fas MoAb-induced apoptosis and activation of SAPK or p38 MAPK in MM cells. Culture of MM cells with IL-6 before treatment with anti-Fas MoAb significantly reduced both DNA fragmentation and activation of SAPK, without altering induction of p38 MAPK activity. These results therefore suggest that anti-Fas MoAb-induced apoptosis in MM cells is associated with activation of SAPK, and that IL-6 may both inhibit apoptosis and modulate SAPK activity.

Description: Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi’s sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P = .01), CD86 (P = .0003), and CD14 (P = .04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.

Description: Hematological malignancies demonstrate susceptibility to T cell mediated killing as evidenced by the potent graft versus disease effect mediated by alloreactive lymphocytes following allogeneic transplantation. A major area of investigation concerns the development of adoptive immunotherapy that would more selectively target and eliminate tumor cells. This strategy is dependent on the expansion of tumor reactive effector cells while minimizing the presence of regulatory T cells and other contributing elements to tumor mediate immunosuppression. Prior studies have demonstrated that ligation of the CD3/CD28 complex results in expansion of T cells ex vivo with phenotypic characteristics that are dependent on the immunologic milieu at the time of stimulation. We have developed a promising cancer vaccine in which autologous tumor cells are fused with dendritic cells (DCs) resulting in the presentation of a wide spectrum of tumor antigens in the context of DC mediated costimulation. We postulated that stimulation with DC/tumor fusions followed by anti-CD3/CD28 would result in the expansion of activated T cells targeting tumor antigens. Tumor cells were obtained from peripheral blood of patients with AML, or bone marrow aspirates of patients with AML and multiple myeloma. DCs were generated from adherent mononuclear cells cultured with rhIL-4, GM-CSF and TNF and fused with tumor cells by coculture in 50% solution of polyethylene glycol. T cells were stimulated by DC/tumor fusions prior to or following exposure to antiCD3/CD28 antibody coated plates for 48 hours. Stimulation by fusions followed by anti-CD3/CD28 resulted in the synergistic expansion of T cells as manifested by the stimulation index (SI) in excess of that seen when cells were stimulated by either pathway alone or first stimulated by anti-CD3/CD28 then fusions. DC/AML fusions induced autologous T cell proliferation with an SI of 3.3 with memory effector cells (CD45RO+) comprising 10% of the total T cell population. In contrast, sequential stimulation with DC/AML fusions followed by anti-CD3/CD28 resulted in a rise in T cell proliferation (SI 8.2) of which 39% of the resultant populations expressed CD45RO. Similarly a rise in CD4+/CD25+ cells was observed following sequential stimulation with DC/AML fusions followed by anti-CD3/CD28 (9.3% vs. 2.7% following stimulation with DC/AML fusions alone). In addition, an increased percentage of CD4+/CD25+ cells expressed IFNγ when exposed to anti-CD3/CD28 following coculture with fusion cells (7% compared to 2% with fusions alone). A significant rise in the percent of Foxp3+ cells was not seen. Expression of granzyme B is up regulated in activated cytolytic CD8+ T cells that confer perforinmediated killing of target cells. As compared to un-stimulated T cells, stimulation with DC/tumor fusions or anti-CD3/CD28 alone resulted in a 3.3 and 3.8 fold increase in CD8+ T cells expressing granzyme B, respectively. In contrast, sequential stimulation with DC/tumor fusions and anti-CD3/CD28 induced a 19-fold expansion of granzyme B+ cells consistent with their enhanced cytolytic capacity. In addition, sequential stimulation with DC/tumor fusions and anti-CD3/CD28 results in heightened capacity to lyse autologous tumor targets as compared to T cells stimulated by fusions alone in a cytotoxicity assay. In spectratyping analysis, T cells undergoing sequential stimulation with DC/tumor fusion cells and anti-CD3CD28 demonstrate greater skewing of CDR3-size usage in the T cell receptor as compared to T cells stimulated by fusions or anti-CD3/CD28 alone consistent with the expansion of a defined clonal population. The pattern of gene expression in T cells stimulated sequentially by DC/AML fusions and anti-CD3CD28 is being assessed by gene arrays to further define the unique nature of this population. In conclusion, we have demonstrated that stimulation of T cells by DC/tumor fusions followed by exposure to anti-CD3/CD28 antibodies results in the expansion of tumor reactive activated T cell populations. A clinical trial evaluating the safety and efficacy of adoptive immunotherapy using T cells generated by sequential stimulation with DC/tumor fusions and anti- CD3CD28 for patients with multiple myeloma following autologous transplantation is planned.

Description: Fusions of cancer cells and dendritic cells (DCs) are effective in the treatment of animal tumor models and patients with metastatic renal carcinoma. In this study, we have fused DCs with mouse 4TOO plasmacytoma cells. The results demonstrate that vaccination of mice with the fusion cells (FC/4TOO) is associated with induction of antitumor humoral and cytotoxic T lymphocyte (CTL) responses. Immunization with FC/4TOO cells protected mice against tumor challenge. In addition, treatment of established multiple myeloma with FC/4TOO cells was associated with prolongation of survival but not with eradication of disease. As interleukin (IL)-12 potentiates the induction of immune responses, recombinant mouse IL-12 was administered with the FC/4TOO vaccine. Treatment of mice with FC/4TOO and IL-12 was associated with increased CTL activity and T-cell proliferation responses. Treatment with FC/4TOO and IL-12 also resulted in eradication of established disease. These findings demonstrate that immunization with FC/4TOO fusion cells and IL-12 potentiates antitumor immunity and the treatment of murine multiple myeloma.

Description: Autologous stem cell transplantation results in tumor cytoreduction and improved disease outcomes in patients with multiple myeloma (MM), but patients ultimately relapse from persistent disease. A promising area of investigation is the development of cancer vaccines that educate host immunity to target and eliminate myeloma cells and can be used to eradicate residual disease following autologous stem cell transplantation. The early post-transplant period is characterized by a transient reversal of tumor mediated tolerance due to the reduction in disease bulk, the depletion of regulatory T cells. We have developed a cancer vaccine model in which DCs are fused to autologous MM cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. We are conducting a study in which patients with MM undergo stem cell transplantation followed by post-transplant vaccination with 3 doses of DC/MM fusions. DCs were generated from adherent mononuclear cells cultured with GM-CSF and IL-4 for 5–7 days and matured with TNFα. DCs strongly expressed costimulatory and maturation markers. Myeloma cells were isolated from bone marrow aspirates and were identified by their expression of CD38, CD138, and/or MUC1. DC and MM cells were fused with polyethylene glycol and fusion cells were quantified by determining the percentage of cells that coexpress unique DC and myeloma antigens. To date, 26 patients have been enrolled. All patients have undergone successful vaccine generation. Mean yield of the DC and myeloma preparations was 171×106 and 70×106 cells, respectively. Mean fusion efficiency was 40% and the mean cell dose generated was 4×106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 88%, 86%, and 78%, respectively. As a measure of their potency as antigen presenting cells, fusion cells potently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 12, 57, 31 for T cells stimulated by myeloma cells, DCs, and fusion cell preparations at an APC: T cell ratio of 1:10. Adverse events judged to be potentially vaccine related were mild, and included injection site reactions, pruritis, myalgias, fever, chills, headache, fatigue and tachycardia. To date 14 patients have completed vaccinations and initial follow up of which 8 have achieved a complete remission and 6 a partial remission. Of note, 4 patients achieved complete remission only after undergoing post-transplant vaccination. We are examining the effect of transplant and vaccination on measures of cellular immunity, antitumor immunity and levels of activated as compared to regulatory T cells. T cell responses to PHA mitogen and tetanus toxoid were transiently depressed post-transplant. Similarly, DTH responses to candida antigen were absent post-transplant in all but 1 patient. In contrast, a significant increase was noted post-transplant in circulating tumor reactive lymphocytes as determined by T cell expression of IFNγ by CD4 and CD8 cells following ex vivo coculture with autologous myeloma cell lysate (Mean percentage of tumor reactive CD8 cells was 0.9 and 11 pre and post-transplant, respectively p=0.01; mean percentage of CD4 cells was 0.7 and 2.7; p=0.02). A further amplification of tumor reactive lymphocytes was seen with vaccination in a subset of patients (mean percentage of CD4 and CD8 tumor reactive T cells was 4.9 and 15, respectively). A decrease in the median levels of circulating regulatory T cells and a relative increase in the ratio of activated (CD4/CD25low)/regulatory (CD4/CD25high) cells was observed following transplantation. This finding suggests that although nonspecific T cell responses are muted in the early posttransplant period, there is a greater capacity to recognize tumor antigens, potentially due to the depletion of regulatory T cells and the decline in tumor mediated immune suppression. In summary, fusion cell vaccination in conjunction with stem cell transplantation was well tolerated, induced anti-tumor immunity and clinical responses in patients with MM. The post-transplant period is characterized by increased levels of activated as compared to regulatory T cells and enhanced levels of T cells with the capacity to respond to myeloma cells. The increase in tumor reactive T cells post-transplant is further amplified following exposure to the DC/MM fusion vaccine.

Description: Introduction: We have pioneered a personalized cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells (DCs) such that a broad array of shared and neo-tumor antigens is presented in the context of DC mediated co-stimulation, limiting the risk of antigen escape. In clinical trials of patients with hematologic malignancies, vaccination with DC/tumor fusions induced an expansion of tumor-specific T cells, and resulted in prolonged remissions in a subset of patients. In the current study, we have developed a novel second generation vaccine, whereby a DC/lymphoma fusion vaccine is presented in the context of a unique biomatrix that expresses high levels of the 41BB costimulatory molecule, to further accentuate T cell activation and prevent the establishment of tumor tolerance. In this study, we demonstrate efficacy of DC/lymphoma fusion cell vaccination in a preclinical lymphoma model, and show enhanced potency of the second-generation vaccine. Methods/Results: We first demonstrated the potency of the DC/tumor fusion vaccine in generating anti-tumor immunity in the A20 lymphoma model. Murine DC/A20 fusions were generated from bone marrow derived mononuclear cells cultured with GM-CSF and IL-4 then fused to syngeneic A20 lymphoma cells. DC/A20 fusion cells effectively induced tumor specific immunity as manifested by potent lysis of A20 T cells in vitro as compared to unstimulated T cells in a standard CTL assay. Consistent with this observation, vaccination with DC/A20 fusions effectively induced lymphoma specific immunity in an immunocompetent murine model. Balb/C mice (30 animals) underwent IV inoculation with 750,000 syngeneic, luciferase and mCherry transduced, A20 cells. 24 hours after tumor cells challenge, 15 mice were treated subcutaneously with 105 DC/A20 fusions. Tumor burden was detected using BLI imaging. 10 days post inoculation, within the untreated cohort all 15/15 mice had detectable tumor whereas within the treated group, 5 mice did not demonstrate any evidence of disease and 5 mice demonstrated minimal disease. We subsequently demonstrated that patient derived autologous DC/lymphoma fusions stimulated T cell mediated lysis of primary lymphoma cells. DC were generated from patient derived peripheral blood mononuclear cells cultured with GM-CSF and IL-4 and matured with TNFa. Primary lymphoma cells were isolated from resected tumor and fused with DC at a ratio of 10:1. Fusion stimulated T cells potently lysed autologous tumor cells as compared to unstimulated T cells (25.7% as compared to 12.66%) in a standard CTL assay. To further enhance vaccine potency, we developed a biomatrix substrate expressing the costimulatory molecule 41BB. Using carbodiimide chemistry we covalently bonded RGD peptide and 41BBL protein to an alginate (Alg)-based scaffold. The Alg/RGD/41BBL scaffold can serve as a supporting microenvironment for the co-culture of T cells and fusion vaccine. We cultured syngeneic T cells with DC/A20 fusion vaccine within a scaffold with or without bound 41BBL and examined the T cells cytotoxicity by a CTL assay as described above. Vaccine mediated stimulation of T cells in the context of the Alg/RGD/41BBL scaffold demonstrated higher levels of tumor lysis as compared to the percent T cells cultured within an Alg/RGD scaffold (22.95% and 13.95% respectively). Conclusion: In the current study we assessed the efficacy of the DC/Lymphoma fusion vaccine to elicit a tumor specific immune response. We succeeded in demonstrating the capacity of DC/Lymphoma fusion vaccine to generate tumor specific T cell cytotoxicity in vitro as well as in vivo in an immunocompetent murine model. Accordingly, we presented patient derived primary tumor results supporting the applicable nature of the DC/Lymphoma vaccine in lymphoma patients. In addition, we developed a second-generation fusion vaccine comprised of the original DC/Tumor vaccine presented to the T cells in an Alg/RGD/41BBL scaffold acting as a nurturing microenvironment for T cell immune specific response against the tumor cells. Our initial results exhibit promising potential and an in vivo experiment with the second-generation fusion vaccine is ongoing. Disclosures Arnason: Celgene/Juno: Consultancy; Regeneron Pharmaceuticals, Inc.: Consultancy. Kufe:Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Hillstream BioPharma: Equity Ownership; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Canbas: Consultancy, Honoraria. Rosenblatt:Dava Oncology: Other: Education; BMS: Research Funding; Partner Tx: Other: Advisory Board; Merck: Other: Advisory Board; Parexel: Consultancy; Imaging Endpoint: Consultancy; Celgene: Research Funding; BMS: Other: Advisory Board ; Amgen: Other: Advisory Board. Avigan:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexel: Consultancy; Takeda: Consultancy.

Description: The epithelial mucin antigen (MUC1) is aberrantly expressed in many epithelial tumors and hematologic malignancies and promotes oncogenesis and tumor progression. MUC1 is recognized by the T cell repertoire and has served as a target for cellular immunotherapy. In the present study, we examined MUC1 as a marker for myeloid leukemia cells and their progenitors and its capacity to serve as a target for leukemia stem cells. Myeloid leukemia cells were isolated from bone marrow aspirates or peripheral blood in patients with high levels of circulating disease. MUC1 was not expressed on unselected leukemia samples (mean expression 3%, n=12). Similarly, low levels of MUC1 expression were seen in leukemic blasts with monocytoid differentiation (mean expression 2.7%, n=5). A subset of leukemia specimens underwent CD34 selection by magnetic bead separation. In contrast to unselected cells, 38% of CD34+ leukemia cells expressed MUC1 (n=5). The leukemia stem cell compartment was isolated by separating CD34+/CD38−/ lineage- fractions by flow cytometric sorting. Leukemia stem cells demonstrated strong expression of MUC-1 by immunohistochemical staining and FACS analysis. Similarly, we examined MUC1 expression on progenitor cells derived from chronic phase chronic myeloid leukemia and following blast transformation. MUC1 was seen in only 4% of CD34+ cells obtained from chronic phase CML samples (n=4) while uniform expression was observed in samples derived from patients with accelerated/blastic phase disease. These data suggest that MUC1 serves as a marker for early leukemia progenitors and is associated with blastic transformation. We assessed the capacity of a cancer vaccine consisting of dendritic cell (DC)/myeloid leukemia fusions to stimulate immune responses that target MUC1 and other antigens expressed by the stem cell compartment. DCs were generated from adherent mononuclear cells that were cultured with GM-CSF and IL-4 and matured with TNFa. DCs were fused with patient derived myeloid leukemia cells using polyethylene glycol as previously described. Fusion cells were quantified by determining the percentage of cells that expressed unique DC and leukemia antigens. DC/AML fusions induced the expansion of MUC1 specific T cells. Stimulation of autologous T cells with DC/AML fusions resulted in a mean 3 fold increase in CD8+ cells binding the MUC-1 tetramer (N=4). DC/AML fusions stimulated anti-tumor immune responses that targeted leukemia stem cells. Fusion stimulated T cells demonstrated increased expression of IFNγ following exposure to lysate generated from unselected leukemia cells (29 fold) and leukemia stem cells (28 fold). In contrast, exposure to renal carcinoma lysate generated only a 5 fold increase in IFNγ. In summary, these findings suggest that leukemic progenitors in AML and accelerated/blast phase CML express MUC-1. DC/tumor fusion vaccines target the MUC-1 protein and the stem cell compartment, and may be a potent immunotherapeutic strategy to eliminate the malignant stem cell clone in AML.

Description: Dendritic cell (DC)-tumor fusions effectively present a broad array of tumor associated antigens in the context of DC derived costimulation. Vaccination with fusion cells induces tumor specific immunity in animal models and clinical studies. We have examined the antigen presenting characteristics of DCs fused with myeloma cells. Immature DCs were generated by culturing adherent mononuclear cells with GM-CSF and IL-4 for 1 week. Maturation was induced by exposure to TNFa for 48 hours. Compared to immature DCs, patient derived mature DC had decreased CD14 expression, increased expression of CD80 and CD83 and increased mean flourescent intensity of CD86. Using polyethyelene glycol (PEG), monocyte derived immature and mature DCs were fused to myeloma cells derived from human cell lines and patient derived bone marrow specimens. Fusion cells were isolated by flow cytometric gating of cells that co-expressed unique DC and tumor antigens. For both immature and mature DC populations, cell fusion was associated with marked upregulation of costimulatory and maturation markers. Mean expression of CD86 was 98% in both fusion cell populations and CD83 was observed in 86% and 84% of immature and mature DC/myeloma fusions, respectively. Immature and mature DC/myeloma fusions prominently express IL-12 (mean 48% and 50%, respectively) and the chemokine receptor, CCR7 (mean 33% and 46%, respectively), necessary for the migration to sites of T cell traffic in the draining lymph node. DC/myeloma fusions induce IFNγ expression by autologous T cells. In 5 serial studies, immature and mature DC/myeloma fusion cells prominently stimulate cytotoxic T lymphocyte mediated lysis of tumor targets (mean 45% and 57%, respectively). Based on these findings we have initiated a clinical trial in which successive cohorts of patients with multiple myeloma undergo vaccination with myeloma cells fused with autologous mature DCs administered in conjunction with GM-CSF. Patients with clinically stable disease who demonstrate at least 20% marrow involvement with plasma cells are potentially eligible. DCs are generated from adherent mononuclear cells collected by leukapheresis that are cultured with GM-CSF, IL-4 and TNFa. Myeloma cells are obtained from short-term culture of bone marrow aspirate specimens. DCs and myeloma cells are co-cultured in the presence of PEG and fusion cells are quantified by identifying cells that co-express DC (CD86, CD83) and myeloma (CD38, CD138) antigens. To date, 6 patients have been enrolled of which 4 have been vaccinated; 3 with 1x10(6) and 1 with 2 x 10(6) fusion cells. Mean yields of DC, tumor, and fusion cells were 7.64 x107, 1.87 x107, and 6.12 x106 cells, respectively. In contrast to myeloma cells, DC and fusion cells preparations prominently induced allogeneic T cell proliferation with mean stimulation indices of 47 and 50, respectively. Toxicities judged to be potentially vaccine related have been mild and include muscle aches/stiffness, transient fever, pruritis, rash and fatigue. Vaccination resulted in the induction of tumor specific immunity as determined by increased percentage of CD4+ and CD8+ T cells expressing IFNγ following exposure to autologous tumor lysate. The effect of vaccination on clinical markers of disease is being monitored.