Novel therapy for therapy-resistant mantle cell lymphoma: multipronged approach with targeting of hedgehog signaling.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.

Optimized adoptive T-cell therapy for the treatment of residual mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm with few patients achieving long-term survival with current treatment regimens. High-dose therapy is effective in reducing the tumor burden; however, patients eventually relapse due to minimal residual disease. Having demonstrated efficacy in other malignancies, the effectiveness of dendritic cell-based immunotherapy for minimal residual MCL was examined. We demonstrated that dendritic cells (DC) primed with MCL antigens stimulated the activation of MCL-specific T cells that recognized and destroyed both MCL cell lines and primary MCL in vitro. In addition, in vivo studies demonstrated that adoptively transferred MCL-specific T cells were able to significantly inhibit tumor growth in mice with minimal residual MCL. Subsequently, when combined with CHOP chemotherapy, adoptive T-cell therapy was able to significantly extend the survival of the mice by further reducing the tumor burden. These results clearly show that MCL-specific cellular immunotherapy is effective in treating minimal residual MCL, paving the way for future clinical studies.

Targeting of sonic hedgehog-GLI signaling: a potential strategy to improve therapy for mantle cell lymphoma.

Mantle cell lymphoma (MCL) has one of the worst clinical outcomes among the B-cell lymphomas, with a median survival of only 3 to 4 years. Therefore, a better understanding of the underlying mechanisms that regulate MCL proliferation/survival is needed to develop an effective therapy. Because sonic hedgehog (Shh)-GLI signaling has been shown to be important in the proliferation and survival of several cancers, and no such information is available for MCL, this study was undertaken. Our results show that the molecules associated with Shh-GLI signaling, such as PTCH and SMO receptors, and GLI1 and GLI2 target transcription factors were expressed in the human MCL cell lines and primary MCL cells from patients. Perturbation of this signaling in the presence of exogenous Shh/cyclopamine significantly (P < 0.001) influenced the proliferation of JVM2 MCL cells. Furthermore, down-regulation of GLI transcription factors using antisense oligonucleotides not only resulted in significantly (P < 0.001) decreased proliferation of the MCL cells but also significantly (P < 0.05) increased their susceptibility to chemotherapeutic drug, doxorubicin. Also, down-regulation of GLI decreased cyclin D1 and BCL2 transcript levels, which suggests that these key molecules might be regulated by GLI in MCL. Thus, our results indicate a significant role for Shh-GLI signaling in the proliferation of MCL, and molecular targeting of GLI is a potential therapeutic approach to improve the treatment for MCL.

Immunotherapy of human neuroblastoma using umbilical cord blood-derived effector cells.

Tumors of the nervous system, including neuroblastoma and glioblastoma, are difficult to treat with current therapies. Despite the advances in cancer therapeutics, the outcomes in these patients remain poor and, therefore, new modalities are required. Recent literature demonstrates that cytotoxic effector cells can effectively kill tumors of the nervous system. In addition, we have previously shown that umbilical cord blood (UCB) contains precursors of antitumor cytotoxic effector cells. Therefore, to evaluate the antitumor potential of UCB-derived effector cells, studies were designed to compare the in vitro and in vivo antitumor effects of UCB- and peripheral blood (PB)-derived antigen-nonspecific and antigen-specific effector cells against tumors of the nervous system. Mononuclear cells (MNCs) from UCB were used to generate both interleukin-2 (IL-2)-activated killer (LAK) cells and tumor-specific cytotoxic T lymphocytes (CTLs). UCB-derived LAK cells showed a significant in vitro cytotoxicity against IMR-32, SK-NMC, and U-87 human neuroblastoma and glioblastoma, respectively. In addition, the CTLs generated using dendritic cells primed with IMR-32 tumor cell lysate showed a selective cytotoxicity in vitro against IMR-32 cells, but not against U-87 or MDA-231 cells. Furthermore, treatment of SCID mice bearing IMR-32 neuroblastoma with tumor-specific CTLs resulted in a significant (p < 0.01) inhibition of tumor growth and increased overall survival. Thus, these results demonstrate the potential of UCB-derived effector cells against human neuroblastoma and warrant further preclinical studies.

Dendritic cell-based therapy for mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a B cell malignancy that is resistant to conventional therapies. High-dose therapy (HDT) followed by stem cell transplantation is effective in inducing remission. However, residual lymphoma cells are eventually responsible for the subsequent relapse. Effective therapeutic strategies to eliminate the residual lymphoma is required. In this study, we have examined the in vitro and in vivo anti-lymphoma effects of MCL-specific cytotoxic T lymphocytes (CTLs) that were generated using dendritic cells (DCs) fused with MCL cells for immunostimulation. Dendritic cells were generated in vitro using dendritic cell-specific medium, cytomorphology, immunophenotypes and functional capabilities of the generated DCs were studied. Such DCs were then used for the preparation of DC-MCL hybrids and the DC-MCL hybrids were used to generate CTLs against MCL cells and tested for their MCL-specific cytotoxicity in vitro and in vivo. The CTLs demonstrated MCL-specific cytotoxicity in vitro against GRANT-519, a human MCL cell line. These CTLs did not show significant effect against an irrelevant target. To test the in vivo therapeutic effect of DC-MCL hybrid-stimulated CTLs, a preclinical model consisting of NOD-SCID mice bearing Granta 519 was developed. The NOD-SCID mice bearing Granta-519 MCL tumors were treated with DC-MCL hybrids and the same donor T lymphocytes. There was an increase in survival (60% in mice treated with DC-MCL hybrid approach compared to 20% in the untreated group). Histological analysis of liver from control and treated mice displayed a decrease in the number of the tumor nodules in the treatment group. These results indicate the potential of DC-based therapy for the treatment of MCL.

Murine mammary adenocarcinoma cells transfected with p53 and/or Flt3L induce antitumor immune responses.

Transfection of tumors with tumor-associated antigens (Ags) or cytokines can increase immunogenicity and slow down tumor growth. However, the effect of cotransfection with genes that encode a tumor-associated Ag, such as the tumor suppressor gene p53, and a cytokine has been rarely investigated. We report that transfection of 4T1 mammary tumor cells (p53-null) with the dendritic cell (DC) growth factor, fms-like tyrosine kinase 3 ligand (Flt3L), significantly delayed their growth in vivo, resulting in the rejection of 100% of the tumors formed by injection of tumor cells cotransfected with Flt3L and p53. Immunization with irradiated 4T1 cells transfected with Flt3L induced DC infiltration of the immunization site and significantly increased the antitumor T-cell responses. Further, immunization with irradiated 4T1 cells cotransfected with p53 and Flt3L significantly increased p53-specific immune responses, as compared to vaccination with 4T1 cells transfected with either Flt3L or p53 alone. These responses included increased activity against clone 66 (Cl-66), a sister tumor to 4T1 with high murine mutant p53 expression levels. Challenge with Cl-66 revealed that immunization with irradiated 4T1-Flt3L-p53 cells significantly slowed growth, prolonged survival, and resulted in complete remissions. Further, immunization with irradiated 4T1-Flt3L also slowed Cl-66 growth, although to a lesser extent than 4T1-Flt3L-p53. We suggest that immunization with DCs transfected with the Flt3L transgene and a tumor Ag may potentially heighten T-cell responses and therapeutic activity.

Fractionation of Aloe vera L. inner gel, purification and molecular profiling of activity.

Products derived from the inner gel of the Aloe vera L. plant have demonstrated multiple clinical activities, and are used routinely to accelerate wound healing. However, typical of natural products, the complex nature of Aloe vera gels may contribute to diverse pharmacologic activities. Our focus on the hematopoietic activities of Aloe vera extracts is extended by these functional studies, which used purified fractions from Aloe vera gel and included a preliminary organ-specific in vitro molecular profile. Studies using a >99% pure carbohydrate fraction from Aloe vera extracts revealed increased hematopoietic and hematologic activity compared to the starting material. In addition, this fraction differentially regulated liver and lung cytokine mRNA levels, resulting in significant increases in message for hematopoietic cytokines [granulocyte colony stimulating factor (G-CSF) and stem cell factor (SCF)]. This profile of activity differed from another fraction obtained from Aloe vera, suggesting the potential for diverse pharmacologic activity. The molecular studies were undertaken using co-cultures of organ slices to limit the amount of purified material required. In summary, these studies revealed significant hematopoietic activity by both pharmacologic and molecular analysis using a >99% pure carbohydrate fraction from Aloe vera gels.

Cytotoxicity of cord blood derived Her2/neu-specific cytotoxic T lymphocytes against human breast cancer in vitro and in vivo.

The Her2/neu oncogene encodes a transmembrane protein with homology to the epidermal growth factor receptor. Overexpression of this gene contributes to the aggressiveness of breast cancer and poor prognosis. Therefore, Her2/neu is an ideal target molecule for generating effective cytotoxic T lymphocytes (CTLs) against breast cancers. This study reports on the generation of Her2/neu-specific CTL from umbilical cord blood mononuclear cells (UCBC) using dendritic cells primed with Her2/neu-derived peptide (KIFGSLAFL, E75) for immunostimulation. The CTLs showed specific cytotoxicity to Her2/neu high expressing MDA-453 but not toward Her2/neu low expressing MDA-231 human breast cancer cells. Similarly generated CTLs stimulated with irrelevant peptide pulsed dendritic cells did not show significant cytotoxicity towards breast cancer targets. The phenotypes of cells in culture showed high percentage of CD3+, CD4+ and CD8+T cells as determined by flow cytometry. However, the antibody mediated blocking assay demonstrated that only HLA-Class I restricted CD8+ cells are involved in the cytotoxicity. Furthermore, in vivo studies showed that treatment of SCID mice bearing MDA-453 tumor with Her2/neu-specific CTLs resulted in significant inhibition of tumor growth compared to untreated tumor bearing control mice. These results demonstrate that human umbilical cord blood mononuclear cells are a good source for generating Her2/neu-specific CTLs against human breast cancer both in vitro and in vivo.

Regional, but not systemic recruitment/expansion of dendritic cells by a pluronic-formulated Flt3-ligand plasmid with vaccine adjuvant activity.

Regional recruitment of dendritic cells (DCs) by the local administration of granulocyte macrophage-colony stimulating factor (GM-CSF) or Flt3-ligand (Flt3L) has vaccine adjuvant activity. However, Flt3L, with its DC growth factor activity, has not been extensively studied as a vaccine adjuvant, particularly as a plasmid vector. We report that the intramuscular (IM) injection of a Flt3L plasmid (pNGVL-hFlex), when formulated in a pluronic carrier (SP1017, Supratek Pharma, Inc., Laval, Que., Canada), recruits DC to the injection site and regional lymph nodes (LNs) and augments immune responses to a p17 HIV plasmid vaccine to a greater extent than the injection of a naked DNA vaccine alone. Following IM administration of pNGVL-hFlex, Flt3L mRNA, Flt3L protein and infiltrating DC accumulate at the injection site. The number of DC in the draining LNs are also significantly increased with the greatest increase observed following injection of 2.5 microg of pNGVL-hFlex formulated in 0.01% SP1017. Flow cytometric studies demonstrate that the LN-infiltrating DC is mainly of the CD11c(+)CD11b(-) phenotype (IL-12 producing). Further, the co-injection of pNGVL3-hFlex and p17 HIV plasmids, formulated in SP1017, significantly increases the immune responses to the plasmid vaccine (pVAX-gag). The co-injection of pVAX-gag and pNGVL3-hFlex, formulated in SP1017, significantly increase delayed-type hypersensitivity responses and the numbers of antigen (Ag)-specific interferon-gamma secreting T cells in the spleen (Enzyme Linked Immune Spot (ELISpot) assay), compared to mice immunized with pVAX-gag formulated in SP1017 alone. We conclude that the IM injection of pNGVL-hFlex with SP1017 can increase the number of DC in draining LN and at the site of injection, thereby providing adjuvant activity for a plasmid vaccine resulting in a significantly increased, Ag-specific T cell response.