GM CSF Topo (A63289)

Multiple myeloma is an incurable plasma cell malignancy. Immunotherapy in myeloma patients had limited success to date. We have previously demonstrated that dendritic cells (DCs) pulsed with autologous Ig Id induced Id-reactive CD8(+) T cells and protection against a myeloma tumor challenge. In this work, we studied the therapeutic efficacy of chemotherapy combined with different formulations of DC-based vaccines in mice bearing large plasma cell tumors. The comparative study demonstrated that s.c. injection of DCs loaded with Id coupled to keyhole limpet hemocyanin, s.c. injection of DCs loaded with irradiated tumor cells, and intratumoral injection of naive DCs were similarly effective in mediating tumor regression and long-term survival. However, whereas the Id-keyhole limpet hemocyanin-DC vaccine was inefficient against myeloma cells that lost expression of the Ig H chain, intratumoral injection of naive DCs and s.c. injection of DCs loaded with irradiated tumor cells were highly effective against cells producing L chains only. This may be of particular importance for patients with L chain myeloma. Given that T cells respond primarily to peptides derived from H chain CDRs, attempts to treat L chain disease with myeloma protein-pulsed DCs may be futile. Vaccination with tumor cell-loaded DCs may, however, induce an effective antitumor response.

Dendritic cells (DCs) have a major role in regulating immune responses, including tumor immunity and peripheral tolerance. In the present study, we identified novel functions of herbal medicines in DCs by screening 99 herbal medicines, most of which are among the 210 Chinese medicines approved by the Ministry of Health, Labour, and Welfare, Japan. Ethanol extracts were prepared, and a murine epidermal-derived Langerhans cell line, XS106, was used to screen the 99 extracts by analyzing major histocompatibility complex (MHC) class II expression. Amomi Semen (amomum seed), Polyporus (polyporus sclerotium), and Plantaginis Semen (plantago seed) potently activated XS106 and were selected for further analysis. The effects of these extracts on bone marrow-derived DCs (BM-DCs) generated in vitro were then analyzed using surface phenotype (MHC class II, CD80, and CD86) and interleukin (IL)-12p70 production as indicators. BM-DCs treated with Amomi Semen extract exhibited activated phenotypes and secreted IL-12p70. The activation level was similar to that induced by lipopolysaccharides. Finally, an E.G7-OVA tumor model (E.L4-OVA transfectant) was used to examine the anti-tumor effects of Amomi Semen extract. Vaccination of mice with a subcutaneous injection of BM-DCs treated with Amomi Semen extract and OVA peptide significantly inhibited the growth of tumor cells and prolonged survival time compared to controls. Furthermore, therapeutic effects were observed on established tumors. The inhibition rates for both the prophylactic and therapeutic protocols were comparable to those of lipopolysaccharides. These results indicate that Amomi Semen extract potently activate DCs and is potentially useful for DC vaccination.

We demonstrated that intranasal granulocyte-macrophage colony-stimulating factor given to immunosuppressed mice infected with pulmonary aspergillosis resulted in a sixfold reduction in the lung fungal burden compared to the result for saline-treated mice (P = 0.045). These data suggest that lung-targeted immunotherapy may be complementary to antifungal agents and may improve patient responses.

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I-bound protein-derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non-glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)-derived peptides O-glycosylated with a Tn epitope (GalNAc) using HLA-A*0201 single chain (HHD)-transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA-A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide-HLA-A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross-reactivity against target cells loaded with glycosylated and non-glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1-HHD and HHD mice and induced effective lysis of (glyco)peptide-loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1-expressing murine cell lines. Moreover, immunization with (glyco)peptide-loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1-derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross-reactivity of the elicited CTLs against the glycosylated and non-glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.

Gene vaccination encounters problems different from those of gene therapy since both a short half-life of the gene and a strong immune response to the gene product are desirable. We have evaluated a DNA vaccine consisting of seven plasmids encoding nine HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8(+) T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. Overall, the results indicate that the intradermal delivery of multigene/multisubtype HIV DNA in combination with recombinant GM-CSF is a safe and efficacious strategy for inducing high levels of specific CD8(+) T cells and unusually high titers of antibodies. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial.