Epidermal growth factor receptor peptide vaccination induces cross-reactive immunity to human EGFR, HER2, and HER3.

Current treatments for tumors expressing epidermal growth factor receptor (EGFR) include anti-EGFR monoclonal antibodies, often used in conjunction with the standard chemotherapy, radiation therapy, or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in many patients, drawbacks include its high cost of treatment and side effects associated with multiple drug infusions. As an alternative to monoclonal antibody treatments, we have focused on peptide-based vaccination to trigger natural anti-tumor antibodies. Here, we demonstrate that peptides based on a region of the EGFR extracellular domain IV break immune tolerance to EGFR and elicit anti-tumor immunity. Mice immunized with isoforms of EGFR peptide p580-598 generated anti-EGFR antibody and T-cell responses. Iso-aspartyl (iso-Asp)-modified EGFR p580 immune sera inhibit in vitro growth of EGFR overexpressing human A431 tumor cells, as well as promote antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies induced by Asp and iso-Asp p580 bound homologous regions of the EGFR family members HER2 and HER3. EGFR p580 immune sera also inhibited the growth of the human tumor cell line MDA-MB-453 that expresses HER2 but not EGFR. Asp and iso-Asp EGFR p580 induced antibodies were also able to inhibit the in vivo growth of EGFR-expressing tumors. These data demonstrate that EGFR peptides from a region of the EGFR extracellular domain IV promote anti-tumor immunity, tumor cell killing, and antibodies that are cross reactive with ErbB family members.

Structural studies of human glioma pathogenesis-related protein 1.

Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Šresolution and that of a Zn2+ complex was refined to 2.2 Šresolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

Expression, purification, crystallization and preliminary X-ray analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1.

Glioma pathogenesis-related protein 1 (GLIPR1) is a member of the CAP superfamily that includes proteins from a wide range of eukaryotic organisms. The biological functions of most CAP proteins, including GLIPR1, are unclear. GLIPR1 is up-regulated in aggressive glioblastomas and contributes to the invasiveness of cultured glioblastoma cells. In contrast, decreased GLIPR1 expression is associated with advanced prostate cancer. Forced GLIPR1 overexpression is pro-apoptotic in prostate cancer cells and is being tested in clinical trials as an experimental prostate-cancer therapy. Human GLIPR1 was expressed as a truncated soluble protein (sGLIPR1), purified and crystallized. Useful X-ray data have been collected to beyond 1.9 Šresolution from a crystal that belonged to the orthorhombic space group P2(1)2(1)2 with average unit-cell parameters a = 85.1, b = 79.5, c = 38.9 Šand either a monomer or dimer in the asymmetric unit.

TLR9-targeted biodegradable nanoparticles as immunization vectors protect against West Nile encephalitis.

Vaccines that activate humoral and cell-mediated immune responses are urgently needed for many infectious agents, including the flaviviruses dengue and West Nile (WN) virus. Vaccine development would be greatly facilitated by a new approach, in which nanoscale modules (Ag, adjuvant, and carrier) are assembled into units that are optimized for stimulating immune responses to a specific pathogen. Toward that goal, we formulated biodegradable nanoparticles loaded with Ag and surface modified with the pathogen-associated molecular pattern CpG oligodeoxynucleotides. We chose to evaluate our construct using a recombinant envelope protein Ag from the WN virus and tested the efficiency of this system in eliciting humoral and cellular responses and providing protection against the live virus. Animals immunized with this system showed robust humoral responses polarized toward Th1 immune responses compared with predominately Th2-biased responses with the adjuvant aluminum hydroxide. Immunization with CpG oligodeoxynucleotide-modified nanoparticles resulted in a greater number of circulating effector T cells and greater activity of Ag-specific lymphocytes than unmodified nanoparticles or aluminum hydroxide. Ultimately, compared with alum, this system offered superior protection in a mouse model of WN virus encephalitis.

Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody.

West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.

A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells.

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.

The multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer.

Although glyceraldehyde-3-phosphate dehydrogenase's (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3' untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDH's association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich-containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression.

West Nile virus envelope protein inhibits dsRNA-induced innate immune responses.

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.

Antibodies targeting linear determinants of the envelope protein protect mice against West Nile virus.

The flavivirus envelope (E) protein mediates cellular attachment and fusion with host cell membranes and is recognized by virus-neutralizing antibodies. We raised antibodies against a broad range of epitopes by immunizing a horse with recombinant West Nile virus (WNV) E protein. To define epitopes recognized by protective antibodies, we selected, by affinity chromatography, immunoglobulins against immobilized linear peptides derived from parts of the E protein. Immunoglobulins binding 9 different peptides from domains I, II, and III of the E protein neutralized WNV in vitro. This indicates that multiple protective epitopes can be found in the E protein. Immunoglobulins recognizing 3 peptides derived from domains I and II of E protein protected mice against a lethal challenge with WNV. These immunoglobulins recognized the E proteins of related flaviviruses, demonstrating that antibodies targeting specific E protein epitopes could be developed for prevention and treatment of multiple flavivirus infections.

Correlation of tumor phenotype with c-fms proto-oncogene expression in an in vivo intraperitoneal model for experimental human breast cancer metastasis.

Although proto-oncogene expression has been shown to correlate with clinical outcome in breast carcinoma, an experimental model has not been proposed to study this phenomenon in vivo. In addition, the ability to modulate this proto-oncogene in vivo to correlate with phenotypic behavior has not been determined. Utilizing an intraperitoneal model for metastatic spread with BT20 human breast carcinoma cells, clonally expanded cells expressing five fold higher c-fms protein were compared with parent BT20 cells as well as an underexpressing clone using intrasplenic injection following left flank cut-down in female nude and Severe combined immunodeficient (SCID) mice. Athymic BALB/c nude and SCID animals were observed for clinical evidence of tumorigenicity with necropsy performed at either 50 or 80 days unless compromised earlier. Immunohistochemistry (IHC) of the harvested tumors was performed to correlate c-fms expression from its original in vitro culture to the in vivo model. At day 50, differences in primary tumor take and spread to the pelvis were already evident favoring the c-fms over-expression group with IHC of these tumors revealing significantly higher intensity of staining for c-fms, (mean H score of 205 vs. 43 in the over-expression and parent groups, respectively). At day 80, tumor take and spread was comparable; however, tumor size in the over-expression group was significantly larger than the parent and under-expressing group in both the BALB/c and SCID experiments. Modulation of c-fms proto-oncogene expression was also achieved using the anti-glucocorticoid, RU-486, via oral administration to SCID mice with subsequent correlation to IHC staining. This model thus provides tumors of significant size and organ diversity which retain their phenotype early in tumorigenesis allowing an early endpoint to assess efficacy of novel treatments.

Drosophila myosin V is required for larval development and spermatid individualization.

Class V myosins are multifunctional molecular motors implicated in vesicular traffic, RNA transport, and mechanochemical coupling of the actin and microtubule-based cytoskeletons. To assess the function of the single myosin V gene in Drosophila (MyoV), we have characterized both deletion and truncation alleles. Mutant animals exhibit no detectable defects during embryogenesis but are delayed in larval development; most die prior to 3rd instar. MyoV protein is widely distributed; however, there are no obvious cytological defects in mutant larval tissues where MyoV was normally highly expressed. Of the few adult MyoV mutant escapers, females were fertile but males were not. We examined the expression of MyoV during spermatogenesis. MyoV was associated with membranes, microtubule, and actin structures required for spermatid maturation; MyoV was strongly associated with the sperm nuclei during the maturation of the actin-rich investment cones that package spermatids in individual membranes. In MyoV mutant escaper males, the early stages of spermatogenesis were normal; however, in the later stages, the investment cones stained weakly for actin and their registration was disrupted; no mature sperm were produced. Our results suggest that MyoV contributes to the formation of the actin-based investment cones and acts to coordinate and/or anchor these structures and other components of the individualization complex.

Glyceraldehyde-3-phosphate dehydrogenase binds to the AU-Rich 3' untranslated region of colony-stimulating factor-1 (CSF-1) messenger RNA in human ovarian cancer cells: possible role in CSF-1 posttranscriptional regulation and tumor phenotype.

The overexpression of the colony-stimulating factor-1(CSF-1) by epithelial ovarian cancer cells enhances invasiveness and metastatic properties, contributing to the poor prognosis of the patients. It has been suggested that CSF-1 3' untranslated region containing AU-rich elements (ARE) could regulate CSF-1 posttranscriptional expression and be responsible for its aberrant abundance in such cancer cells. In this study, normal (NOSE.1) and malignant (Hey) ovarian epithelial cells were used to examine CSF-1 expression and regulation. CSF-1 overexpression in Hey cells was found to associate with increased invasiveness, motility, urokinase activity, and virulence of tumorigenicity, compared with NOSE.1 cells, which expressed little CSF-1. CSF-1 ARE was further found to serve as an mRNA decay element that correlates with down-regulation of protein translation. Moreover, such down-regulation was found more prominent in NOSE.1 than in Hey cells, suggesting differences in posttranscriptional regulation. As a variety of trans-acting factors [AU-binding protein (AUBP)] are known to modulate messenger stability through binding to such elements, we examined the protein content of both cell lines for their ability to bind the CSF-1 ARE. Our results strongly suggested the abundance of such AUBP activity in Hey cells. We isolated a 37-kDa AUBP, which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To summarize, our study identified GAPDH as an AUBP abundant in Hey cells, where it binds to CSF-1 ARE that imparts mRNA decay. These data suggest that GAPDH binding to CSF-1 ARE sequence prevents CSF-1 mRNA decay and subsequent down-regulation of CSF-1 protein translation, leading to CSF-1 overexpression and increased metastatic properties seen in ovarian cancer.

SGK1, a potential regulator of c-fms related breast cancer aggressiveness.

The aggressive behavior of breast cancer cells can at times be modulated by hormonal mechanisms. Exposure to glucocorticoids (GC) has been shown to stimulate the invasiveness, motility and adhesiveness of breast cancer cells containing the glucocorticoid receptor. This is largely explained by GC-associated overexpression of the c-fms proto-oncogene, which encodes the receptor for the colony stimulating factor-1 (CSF-1). Our objective is to investigate additional GC-associated genetic alterations that could modulate c-fms related malignant behavior in breast cancer cells. A microarray technique using an oligonucleotide array representing 16,700 known expressed human genes was used to analyze the gene expression profile of breast cancer cells exposed to dexamethasone (Dex) or vehicle. Results were confirmed by western blot analysis. Six genes were found to be consistently differentially overexpressed in the Dex-exposed cells compared to control. We focused on serum-glucose kinase 1 (SGK1), a serine-threonine kinase known to be involved in intracellular signal transduction pathways and induced by GC and serum. An adhesion assay was performed on extracellular matrix after exposing the breast cancer cells to Dex, CSF-1 or to Dex or CSF-1 plus LY294002, a functional inhibitor of SGK1 action. Exposure to LY294002 significantly decreased both CSF-1 and Dex-induced adhesiveness to the level of control cells. SGK1 may act as a downstream intracellular regulator of c-fms, particularly of c-fms-induced adhesiveness of breast cancer cells after exposure to GC or CSF-1. This finding may have implications for potential therapeutic interventions aimed at decreasing the aggressiveness of breast cancer cells.