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Provided are adenoviral vectors for generating an immune response to antigen. The vectors comprise a transcription unit encoding a secretable polypeptide, the polypeptide comprising a secretory signal sequence upstream of a tumor antigen upstream of CD40 ligand, which is missing all or substantially all of the transmembrane domain rendering CD40L secretable. Also provided are methods of generating an immune response against cells expressing a tumor antigen by administering an effective amount of the invention vector. Further provided are methods of generating an immune response against cancer expressing a tumor antigen in an individual by administering an effective amount of the invention vector. Still further provided are methods of generating immunity to infection by human papilloma virus (HPV) by administering an effective amount of the invention vector which encodes the E6 or E7 protein of HPV. The immunity generated is long term.
1. A pharmaceutical composition comprising a viral expression vector for generating an immune response against a human tumor antigen, said vector comprising a transcription unit encoding a polypeptide, said polypeptide comprising from the amino terminus a secretory signal sequence, a tumor antigen, and a CD40 ligand, wherein said tumor antigen is different from said CD40 ligand and wherein the tumor antigen is connected to the amino terminus of said CD40 ligand, and wherein said CD40 ligand is missing all or substantially all of the transmembrane domain rendering the CD40 ligand secretable, and wherein said polypeptide is adapted to cause induction in a human of expression of activation molecules CD80 and CD54 on dendritic cells to promote Class I MHC restricted cytotoxic T cell lymphocytes (CTLs).
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4. A pharmaceutical composition comprising a viral expression vector for generating an immune response against a human tumor antigen, said vector comprising a transcription unit encoding a polypeptide, said polypeptide comprising from the amino terminus a secretory signal sequence, a tumor antigen, and a CD40 ligand, wherein said tumor antigen is different from said CD40 ligand and wherein the tumor antigen is connected to the amino terminus of said CD40 ligand, and wherein said CD40 ligand is missing all or substantially all of the transmembrane domain rendering the CD40 ligand secretable, and wherein said polypeptide is adapted to cause dendritic cells in a human to elicit activation and expression of co-stimulatory molecules to promote Class I MHC restricted cytotoxic T cell lymphocytes (CTLs).
5. The pharmaceutical composition of
6. A pharmaceutical composition comprising a viral expression vector for generating cellular and humoral immunity against an infection, said vector comprising a transcription unit encoding a polypeptide, said polypeptide comprising from the amino terminus a secretory signal sequence, an antigen, and a CD40 ligand, wherein said antigen is connected to the amino terminus of said CD40 ligand and is different from said CD40 ligand, and wherein said CD40 ligand is missing all or substantially all of the transmembrane domain rendering the CD40L secretable, and wherein said polypeptide is adapted to generate immunity in a human against the antigen which is part of the infection by promoting Class I and Class II MHC restricted cytotoxic T cell lymphocytes (CTLs).
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This application is a continuation of U.S. patent application Ser. No. 10/534,605, filed on May 11, 2005, now U.S. Pat. No. 8,119,117, which is a national phase application of International Application No. PCT/US2003/036237, filed on Nov. 12, 2003, which claims priority to U.S. Provisional Patent Application Ser. No. 60/425,286 filed on Nov. 12, 2002, the disclosures of which are all hereby incorporated herein by reference.
The present invention relates to the development of immunity against antigen expressing cells using an adenoviral vector that expresses the antigen fused to a secretable form of CD40 ligand.
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
The activation of antigen presenting cells (APCs) including the dendritic cells (DCs), followed by loading of the antigen presenting cell with relevant antigens, is a requisite step in the generation of a T cell dependent immune response against cancer cells. Once activated and loaded with tumor antigens, DCs migrate to regional lymph nodes (LNs) to present antigens (ags) to T cells. Very commonly, these APCs express insufficient amounts of surface activation molecules (1) which are required for optimal activation and expansion of T cell clones competent to recognize tumor antigens. Antigen (ag) presentation to naive T cells, in the absence of costimulatory molecule expression on the surface of the APC, leads to anergy of T cells (2). Moreover, cross-presentation by DCs without CD4+ T cell help also results in peripheral deletion of Ag-specific cells in regional LNs (3). In contrast, in the presence of CD4+ T cell help, DCs change their functional ability to cross-prime cells, resulting in clonal expansion of effector T cells (4). This CD4+ T cell help can be replaced with CD40-CD40 ligand (CD40L) interactions (5). CD40L is a 33-kDa type II membrane protein and a member of the TNF gene family which is transiently expressed on CD4+ T cells after TCR engagement (6).
The ability of DCs to generate anti-tumor immune responses in vivo has been documented in a number of animal tumor models (7, 8). However, DC-mediated induction of immunity represents a major therapeutic challenge. The current procedures used for isolating and activating DCs are resource intensive and are difficult to apply to routine clinical practice. In addition, it is difficult to ensure that the antigen presenting cells express appropriate adhesion molecules and chemokine receptors to attract the these DCs to secondary lymphoid organs for priming T cells (9-14).
In one aspect, the present invention provides an adenoviral expression vector for generating immunity against antigen. The vector comprises a transcription unit encoding a polypeptide comprising, from the amino terminus, a secretory signal sequence upstream of a tumor antigen upstream of CD40 ligand, which is missing all or substantially all of the transmembrane domain, rendering CD40 ligand secretable. The secretory signal sequence functions to direct the tumor antigen-CD40 ligand fusion protein to compartments of the cell which cause the fusion protein to be secreted from the cell.
In one embodiment, the tumor antigen is a human tumor antigen. In another embodiment, the human tumor antigen is the E7 protein of human papilloma virus (HPV).
In some embodiments, the transcription unit includes sequence that encodes a linker between the tumor antigen and the CD40 ligand. Suitable linkers may vary in length and composition.
In another embodiment, the adenoviral expression vector comprises a human cytomegalovirus promoter/enhancer for controlling transcription of the transcription unit.
In still another embodiment, the CD40 ligand is human CD40 ligand.
In yet another embodiment, the CD40 ligand which lacks a functional transmembrane domain is one which contains less than 10% of the transmembrane domain or does not contain a transmembrane domain.
In another aspect, the present invention provides methods of generating an immune response in an individual against cells expressing a tumor antigen by administering an effective amount of the invention vector which encodes the tumor antigen.
In one embodiment, the tumor antigen is a human tumor antigen. In another embodiment, the human tumor antigen is the E7 protein of human papilloma virus.
In yet another embodiment, the cells are cancer cells. In another embodiment, the cancer cells are cervical cancer cells.
In still another embodiment, the method results in the generation of cytotoxic CD8+ T cells against said tumor associated antigen. In another embodiment, the vector following administration is taken up by cells which subsequently secrete a fusion protein encoded by the transcription unit.
In yet another aspect, the present invention provides methods of treating an individual with cancer that expresses a tumor antigen. The method comprises administering to the individual an effective amount of the invention expression vector which encodes the tumor antigen.
In one embodiment, the tumor antigen is a human tumor antigen. In another embodiment, the human tumor antigen is the E7 protein of human papilloma virus.
In yet another embodiment, the cells are cancer cells. In another embodiment, the cancer cells are cervical cancer cells.
In still another embodiment, the method results in the generation of cytotoxic CD8+ T cells against said tumor associated antigen. In another embodiment, the vector following administration is taken up by cells which subsequently secrete a fusion protein encoded by the transcription unit.
In a further aspect, the present invention provides method of generating immunity in a subject to infection by human papilloma virus, comprising administering to the individual an effective amount of the invention adenoviral expression vector wherein the tumor antigen is the E6 or E7 protein of human papilloma virus.
In the above methods, the vector is advantageously administered subcutaneously and may be given on subsequent time to increase the immune response. The immunity against the tumor antigen expressing cells is long lasting.
In order to improve DC activation and tumor antigen loading, an immunization strategy was developed that used a recombinant adenovirus encoding a secretable form of the oncoprotein E7 from high-risk human papilloma viruses (HPV) fused to CD40 ligand from which the transmembrane domain had been deleted. This construct was engineered to be secretable by deleting the cytoplasmic and transmembrane domains from CD40 ligand, which was located downstream from E7 encoding DNA and DNA encoding a signal sequence.
The term "adenoviral expression vector" as used herein, refers to any adenoviral vector that includes exogenous DNA inserted into its genome which encodes a polypeptide. The vector must be capable of replicating and being packaged when any deficient essential genes are provided in trans. An adenoviral vector desirably contains at least a portion of each terminal repeat required to support the replication of the viral DNA, preferably at least about 90% of the full ITR sequence, and the DNA required to encapsidate the genome into a viral capsid. Many suitable adenoviral vectors have been described in the art. U.S. Pat. No. 6,440,944; see U.S. Pat. No. 6,040,174 (replication defective E1 deleted vectors and specialized packaging cell lines. A preferred adenoviral expression vector is one that is replication defective in normal cells.
The term "transcription unit" as it is used herein in connection with the adenoviral expression vector means a stretch of DNA that is transcribed as a single, continuous mRNA strand by RNA polymerase, and includes the signals for initiation and termination of transcription. For example, a transcription unit of the invention is nucleic acid that encodes from 5′ to 3′ a secretory signal sequence, the E7 protein from HPV and CD40 ligand without its transmembrane domain. The transcription unit is in operable linkage with transcriptional and/or translational expression control elements such as a promoter and optionally any upstream or downstream enhancer element(s). A useful promoter/enhancer is the cytomegalovirus (CMV) immediate-early promoter/enhancer (see U.S. Pat. Nos. 5,849,522 and 6,218,140).
The term "secretory signal sequence" (aka "signal sequence," "signal peptide," leader sequence," or leader peptide") as used herein refers to a short peptide sequence, generally hydrophobic in charter, comprising about 20 to 30 amino acids which is synthesized at the N-terminus of a polypeptide and directs the polypeptide to the endoplasmic reticulum. The secretory signal sequence is generally cleaved upon translocation of the polypeptide into the endoplasmic reticulum. Eukaryotic secretory signal sequences are preferred for directing secretion of the exogenous gene product of the adenoviral expression vector. A variety of suitable such sequences are well known in the art and include the secretory signal sequence of human growth hormone, immunoglobulin kappa chain, and the like.
The term "tumor associated antigen" (TAA) as it is used herein refers to a protein which is present on tumor cells, and on normal cells during fetal life (onco-fetal antigens), after birth in selected organs, or on many normal cells, but at much lower concentration than on tumor cells. A variety of TAA have been described. In contrast, tumor specific antigen (TSA) (aka "tumor-specific transplantation antigen or TSTA) refers to a protein not present on non-tumor cells. TSA usually appear when an infecting virus has caused the cell to become immortal and to express virus antigens. TSAs not induced by viruses can be idiotypes of the immunoglobulin on B cell antigens (TAA) are more common than TSA.
Both TAA and TSA may be the immunological target of an adenoviral expression vector vaccine of the present invention. Unless indicated otherwise, the term "tumor antigen" is used herein to refer collectively to TAA and TSA. A preferred tumor antigen is the E6 or E7 protein of HPV. These antigens are preferably from HPV type 16.
The term "linker" as it used herein with respect to the transcription unit of the adenoviral vector refers to one or more amino acid residues between the carboxy terminal end of the tumor antigen and the amino terminal end of the CD40 ligand (lacking a functional transmembrane domain). The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. The linker is generally from about 3 to about 15 amino acids long, more preferably about 5 to about 10 amino acids long, however, longer or shorter linkers may be used or the linker may be dispensed with entirely.
The term "CD40 ligand" as used herein refers to a type II membrane polypeptide having an extracellular or cytoplasmic domain at its N-terminus, a transmembrane region and an extracellular domain at its C-terminus. CD40L is a member of the tumor necrosis factor superfamily of molecules and carries the designation TNF5. Unless otherwise indicated the full length CD40L is designated herein as "CD40L," "wtCD40L" or "wtTmCD40L." The form of CD40L where the cytoplasmic domain has been deleted is designated herein as "ΔCtCD40L." The form of CD40L where the transmembrane domain has been deleted is designated herein as "ΔTmCD40L." The form of CD40L where both the cytoplasmic and transmembrane domains have been deleted is designated herein as "ΔCtΔTmCD40L." The nucleotide and amino acid sequence of CD40L from mouse and human is well known in the art and can be found, for example, in U.S. Pat. No. 5,962,406 (Armitage et al.).
Murine CD40L (mCD40L) is 260 amino acids in length. The cytoplasmic (Ct) domain of mCD40L extends approximately from position 1-22, the transmembrane domain extends approximately from position 23-46, while the extracellular domain extends approximately from position 47-260.
Human CD40L (hCD40L) is 261 amino acids in length. The cytoplasmic domain of hCD40L extends approximately from position 1-22, the transmembrane domain extends approximately from position 23-46, while the extracellular domain extends approximately from position 47-261.
The phrase "CD40 ligand is missing all or substantially all of the transmembrane domain rendering CD40 ligand secretable" as it is used herein refers to a recombinant form of CD40 ligand that can be secreted from a cell. The transmembrane domain of CD40L containing about 24 amino acids in length functions to anchor CD40 ligand in the cell membrane. CD40L from which all of the transmembrane domain has been deleted is CD40 ligand lacking residues 23-46. CD40 ligand missing substantially all of the transmembrane is one that contains 6 residues or less of sequence at one end of the transmembrane domain, more preferably less than about 4 residues of sequence at one end of the transmembrane domain, even more preferably less than about 2 residues of sequence on one end of the transmembrane domain, and most preferably 1 residue or less on one end of the transmembrane domain. Thus, a CD40L that lacks substantially all of the transmembrane domain rendering the CD40L secretable is one that contains no more than six residues of sequence on one end of the domain. Such as CD40L would contain, in addition to the extracellular domain and optionally the cytoplasmic domain, no more than amino acids 41-46 or 23-28 located in the transmembrane domain of CD40L.
It should be understood that a CD40L which lacks a functional transmembrane domain may still include all or a portion of the cytoplasmic domain. Likewise, a CD40L which lacks a functional transmembrane domain may include all or a substantial portion of the extracellular domain.
As used herein, an adenoviral vector of the present invention can be administered as a vaccine to induce immunity to a tumor associated antigen. The viral vector may be formulated as appropriate with a pharmaceutically acceptable carrier. Accordingly, the viral vectors may be used in the manufacture of a medicament or pharmaceutical composition. Viral vectors of the invention may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. Liquid formulations may be buffered, isotonic, aqueous solutions. Powders also may be sprayed in dry form. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water, or buffered sodium or ammonium acetate solution. Such formulations are especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol sodium chloride, sodium citrate, and the like.
Alternately, viral vectors may be prepared for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the viral vectors. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, or an aqueous or non-aqueous suspension.
Viral vectors of the invention may be formulated to include other medically useful drugs or biological agents. The viral vectors also may be administered in conjunction with the administration of other drugs or biological agents useful for the disease or condition that the invention compounds are directed.
As employed herein, the phrase "an effective amount," refers to a dose sufficient to provide concentrations high enough to generate an immune response in the recipient thereof. The specific effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound, the route of administration, the rate of clearance of the viral vectors, the duration of treatment, the drugs used in combination or coincident with the viral vectors, the age, body weight, sex, diet, and general health of the subject, and like factors well known in the medical arts and sciences. Various general considerations taken into account in determining the "therapeutically effective amount" are known to those of skill in the art and are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed, Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990. For administration of adenoviral vectors, the range of particles per administration typically from about 1�107 to 1�1011, more preferably 1�108 to 5�1010, and even more preferably 5�108 to 2�1010. A viral vector can be administered parenterally, such as intravascularly, intravenously, intraarterially, intramuscularly, subcutaneously, or the like. Administration can also be orally, nasally, rectally, transdermally or inhalationally via an aerosol. The viral vectors may be administered as a bolus, or slowly infused. The vector is preferably administered subcutaneously.
The E7 protein derived from the human papillomaviruses (HPVs) was chosen as the exemplary TAA since it has been shown to be a strong stimulus of the cellular immune response, and to be expressed on every cell of a tumor population which is generated from HPV transformed tissue. HPV can cause a variety of epithelial lesions of the skin and genital tract. HPV related diseases of the genital tract constitute the second leading cause of cancer death among women in the world. These include genital warts, cervical intraepithelial neoplasia (CIN) and cancer of the cervix. The HPV type most commonly associated with high grade CIN and cervical cancer is HPV type 16. The majority of cervical cancers express the non-structural HPV16-derived gene products E6 and E7 oncoproteins. In HPV-induced cervical cancer model, the E6/E7 oncoproteins are required for maintenance of the malignant phenotype and their expression correlates with the transforming potential of HPV16 (15-16). Therefore, E6 and E7 represent a target of choice for the therapeutic vaccination (17-19).
The results herein show that the subcutaneous injection of this vector leads to; 1) Local infection of cells surrounding the needle tract; 2) Secretion of the chimeric E7/CD40L transmembrane less fusion protein over a 10 day period from the vector injected cells; 3) Binding of the fusion protein to APCs leading to activation of the APC's and E7 presentation; 4) Migration of the loaded and activated APCs to regional lymph nodes, and 5) Evolution of T cell dependent systemic immunity to cell lines carrying the tumor associated antigen which extends for greater than one year.
Generation of cytotoxic T cell lymphocytes specific for individual populations of tumor cells by antigen presenting cells (dendritic cells) depends on the activation of expression of co-stimulatory molecules. One of the mechanisms through which this can occur is the binding of the CD40 ligand on the plasma membrane of CD4 Helper T Cell lymphocytes to the CD40 receptor on the dendritic cells. Following this activation, dendritic cells migrate to region lymph nodes or to the spleen in which the presentation of the peptides derived from tumor associated antigens induces the activation and expansion of CD8 cytotoxic T cell lymphocyte clones which are competent to recognize the tumor associated antigen.
The evolution of such CD8 cytotoxic T cell lymphocytes, which can recognize tumor associated antigens on tumor cells, which leads to the killing of the tumor cells bearing these antigens, is limited by many factors. One factor is the low level of transfer of the tumor associated antigens from tumor cells into the antigen presenting cells. Another is the failure to induce the co-stimulatory molecules on the antigen presenting cells which are necessary for sustained activation and expansion of tumor antigen specific CD8 cytotoxic T cell lymphocytes which are competent to recognize and kill the tumor cells.
Antibodies which bind to the CD40 receptor on antigen presenting cells can activate the expression of the co-stimulatory molecules in the antigen presenting cells. In vitro infection of the antigen presenting cells (either dendritic cells or tumor cells themselves) with an adenoviral vector (Ad-wtTmCD40L) which carries a CD40 ligand transcription unit has also been used to activate co-stimulatory molecule expression on the antigen presenting cells or the tumor cells (crystal). Injection of these dendritic cells into the tumor nodules or injection of the irradiated Ad-wtTmCD40L infected tumor cells into immunocompetent mouse hosts carrying tumor nodules can lead to regression of the tumor nodules and to increased survival.
In addition, in vivo generation of tumor antigen specific CD8 cytotoxic T cell lymphocytes results from in vitro "loading" of the antigen presenting cells with tumor associated antigens by incubation with tumor antigen peptide fragments, or with the tumor cells themselves, or with adenoviral vectors which carry transcription units encoding the tumor associated antigens, followed by injection of the antigen loaded antigen presenting cells.
In order to develop a more efficient way of generating a tumor antigen CD8 cytotoxic T cell lymphocyte dependent systemic immune response to tumor associated antigens, it was hypothesized that the continuous release of a protein in the subcutaneous space of immunocompetent animals, which was capable of both activating the expression of co-stimulatory molecules in antigen presenting cells and at the same time delivering tumor associated antigen into the intracellular space of antigen presenting cells over a 10 day period, could lead to a very strong CD8 cytotoxic T cell lymphocyte response against the tumor cells bearing the tumor associated antigen.
The fusion protein transcription unit that was chosen to test this hypothesis comprised a tumor associated antigen (the human papilloma virus E7 transforming protein) linked to the amino-terminal end of the CD40 ligand. This protein was engineered to be secreted from cells in which it was being made by deleting the transmembrane and cytoplasmic domains of the CD40 ligand, and attaching a human growth hormone signaling domain to the amino-terminal end of the E7 tumor antigen portion of the fusion protein gene. Subcutaneous injection of an adenoviral vector which carried a transcription unit encoding this secretory form of the tumor associated antigen/CD40 ligand fusion protein (Ad E7ΔCtΔTmCD40L) was chosen to generate adenoviral infected cells in the subcutaneous space of a subject.
It was reasoned that the cells infected in the vicinity of the site of subcutaneous injection of the vector, would release the tumor antigen/CD40 ligand secretory protein for up to 10-14 days (the time period during which most adenoviral vector infected cells produce the protein product of the transgene). Antigen presenting cells (e.g. DC) in the vicinity of the infected cells at the injection site would take up E7 tumor associated antigen, which would be digested in the proteosome with the resultant E7 peptides trafficking to the endoplasmic reticulum where they would bind to Class I MHC molecules. Eventually, the DCs would present the E7 tumor associated antigen on the surface in the Class I MHC molecule. Activated, tumor antigen-loaded antigen presenting cells would migrate to lymphocyte bearing secondary organs such as the regional lymph nodes or the spleen. During the two weeks of continuous release of the tumor antigen/CD40 ligand fusion secretory protein, CD8 cytotoxic T cell lymphocytes which were competent to recognize and kill cells, which carried the tumor associated antigens, would be expanded in the lymph nodes and spleen by the presence of the activated and antigen loaded dendritic cells. The continuous nature of the stimulation and the expansion of the tumor antigen specific cytotoxic T cells by the continuous release from the vector infected cells was believed to generate an immune response which would be greater in magnitude than were possible using a vector which carried a tumor associated antigen/CD40 ligand which was non-secretory. An outline of the events that would occur during the release of secretable form of the E7/CD40L are shown in
The results of the experiments described herein were designed to test these hypotheses and confirmed all the predictions. Subcutaneous injection of a replication incompetent adenoviral vector, which carries a secretory form of the E7 tumor associated antigen/CD40 ligand protein, induced a very robust and long lasting CD8 cytotoxic T cell lymphocyte dependent systemic immune response against cancer cells which carry the E7 tumor associated antigen. Indeed, the experimental results showed that the vector expresses the secretory form of the tumor associated antigen/CD40 ligand fusion protein generated an immune response which is much stronger than the subcutaneous injection of a vector that expresses the non-secretory form of the E7 tumor associated antigen/CD40 ligand fusion protein. It is clear from the these experimental results that the subcutaneous injection of vectors encoding the tumor associated antigen/CD40 ligand secretory fusion protein produces an immune response that is much stronger than occurs following the subcutaneous injection of the vector which carries transcription units encoding only for the E7 tumor associated antigen or the CD40 ligand (secretory or non-secretory).
In addition, it was shown that the local release of the fusion protein generates activation of antigen presenting cells, and their migration to the lymphoid bearing secondary organs. Finally, it was demonstrated herein that the immunological resistance generated by the subcutaneous injection of adenoviral vectors carrying the E7 tumor associated antigen/CD40 ligand secretory transcription unit was specific to cells carrying the tumor associated antigen and was long lasting for up to one year after the vector injection. Although not wishing to be bound by any theory, it is believed that the invention method of linking the tumor associated antigen to a secreted form of CD40 ligand creates a protein which can at the same time both activate antigen presenting cells and antigen load them in vivo avoiding the expense and the logistical problems associated with in vitro generation of antigen presenting cells or cytotoxic T cell lymphocytes.
A second advantage is that the use of the secreted tumor associated antigen/CD40 ligand fusion protein for activation and antigen loading may result in selective binding to the antigen presenting cells and transport of the antigen into the intracellular space of the antigen presenting cell. This would solve the problem of delivering sufficient tumor antigens into the intracellular space of a subject which is frequented by antigen presenting cells so as a sufficient density of tumor associated peptide on the plasma membrane of the antigen presenting cells for generating a strong cytotoxic T cell lymphocyte response against the tumor associated antigen expressing tumor cell.
A third advantage is that the presence of the CD40 ligand in the secreted protein may activate co-stimulatory antigen expression on the antigen presenting cell, thus making the activation independent of initial CD4 T cell lymphocyte help.
A fourth advantage of the secretory nature of the viral fusion protein transcription unit is that there may be continuous release of the activating and antigen loading fusion protein in the space populated by antigen presenting cells over a 10 day period. This may account in part for the robustness of the immune response observed and its durability.
The decision to utilize the HPV E7 protein as the tumor associated antigen of the fusion protein was motivated by several factors. The first is that the E7 protein has been shown to be transforming and essential for the continued proliferation of HPV transformed cells. This property of E7 ensures that most if not all of the cells in the population would carry the antigen. In addition, the choice of a DNA virus transforming protein as the tumor associated antigen was designed to make the experiments unambiguous with respect to the testing of the value of the secretory fusion transcription unit. A separate but important reason for selecting HPV E7 as the tumor associated antigen in the invention vector was to evaluate the ability of E7 to prevent the infection and proliferation of HPV infected cervical epithelial. If such vector could generate an immune response against HPV infected cells, then individuals in whom such a reaction would be generated would be immune and resistant to the infection of the human papilloma virus.
Human papillomavirus (HPV) infection induces a natural host immune response that is not sufficient to eliminate infection. One reason for this is that there are over 100 genotypes of HPV each of which may have a unique amino acid sequence and therefore unique epitopes that allow the virus to escape detection by individuals exposed to other HPV genotypes. A second reason is that there are a small number of individuals who are unable to mount a cellular immune response to HPV infected cells. Such individuals may be the ones in whom chronic HPV infections develop which lead to cervical dysplasia and to invasive cervical cancer. In addition, 60% of human cervical cancers are related to infections with the HPV 16 genotype. Up to 95% of cervical cancer cases are attributable to HPV infections. The second leading cause of cancer death among women worldwide is HPV associated malignancies. In emerging nations of the world, HPV associated malignancies represent the leading cause of death among women between the ages of 25 and 35.
In order to amplify an immune response to the HPV infected cells so as to develop immunity to HPV infection, several methods have been attempted. The therapeutic peptide and protein-based vaccines (15), DNA (16) and viral vector-based vaccines (17), modified tumor cell (18) and dendritic cell-based vaccines (19) all have been previously investigated and reported to generate immune responses to human HPV-associated neoplasms in animal models. These previous studies have shown that a variety of preparation of DCs can stimulate an effective antitumor immunity, including DCs loaded with proteins, DCs fused with tumor cells and DCs transduced with tumor-derived RNA or viral vectors (20, 21, 22). At present, in vitro CD40 ligand activation of MART-1 gene modified DC can promote CD8 T cell lymphocyte mediated immunity to melanoma cells (23). Injection of CD40 ligand-transduced tumor showed a therapeutic effect against the established MCA205 brain tumor cell line (24). These studies indicated that DCs that were loaded with antigen and activated at the same time can provide strong protective immunity.
Recombinant adenoviral vectors offer a potentially superior approach that allows a higher efficiency of gene transfer than that of DNA vaccines. Adenoviral vectors encoding tumor associated antigens also can induce the protective cellular and humoral immunity against such antigens, including those to which tolerance had developed.
Replication-defective adenoviral vectors, used herein to generate continual local release of a fusion protein composed of a tumor antigen (E7) linked to a secretory form of CD40 ligand, facilitated DCs maturation, promoting the development of effective antigen-specific immunity. It was demonstrated that secretable E7-ΔCtΔTmCD40L construction dramatically enhances the potency of the cellular immune response to E7 positive tumor cells. Subcutaneous injection of the Ad E7-ΔCtΔTmCD40L vector elicited strong E7-specific CD8+ T cell-mediated immunity, which could prevent the engraftment of cancer cells which express the E7 tumor associated antigen.
Although not wishing to be bound by any theory, a potential mechanism for the observed enhancement of E7-specific CD8+ T-cell activity following injection of the vector encoding the tumor antigen/secretable CD40L is the enhanced activation and migration of dermal professional APCs which are generated by the subcutaneous release of fusion protein from vector infected cells. In contrast to the strategy of loading DCs with antigen by administering the antigen, secretion of the E7-ΔCtΔTmCD40L protein, by the dermal fibroblasts or other cells in the subcutaneous space may more effectively activate and load local APCs, which may be effective in activating systemic T-cell immunity.
Another factor potentially affecting enhancement of antigen-specific CD8+ T cell activity by the AdE7-ΔCtΔTmCD40L vector is the use of the full-length E7 protein as the tumor associated antigen. Some workers have compared immune responses achieved with vaccines based on either HPV cytolytic T lymphocyte (CTL) plus helper T lymphocyte epitopes or the CTL epitope alone derived from the E7 peptide of the HPV 16 strain The results showed that the E7-specific CD8+ response was significantly greater for the vaccine based on HPV epitopes plus helper Th epitopes than for the vaccine based only on the CTL epitope (15).
In summary, the findings illustrate that linkage of ΔTmCD40L, which is a secretory form of a CD40L, to a tumor associated antigen, can lead to enhanced antigen-specific CD8+ T-cell activity in vivo.
The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.
The transcription unit, E7-ΔCtΔTmCD40L, of the adenoviral vector encoded a signal secretory sequence followed by the HPV type 16 E7 gene which was connected via a linker to the fragment of the CD40 ligand which contained the extracellular domain without the transmembrane or cytoplasmic domains. E7 was small enough that it did not disrupt the natural assembly of a homotrimeric array trimeric of the native wild type CD40 ligand. The fusion protein was engineered to be secreted from vector infected cells by the addition of a signal sequence to the amino-terminal end of the E7 protein and by deletion of the transmembrane and cytoplasmic domains of the CD40 ligand.
The transcription unit was introduced into the E1 gene region of the adenoviral vector backbone. After the adenoviral vector particles were generated in HEK 293 cells, the vector DNA was purified by cesium chloride gradient centrifugation. The presence of the signal peptide in the adenoviral vector was confirmed by restriction enzyme analysis and by DNA sequencing.
Adenoviral vectors with different transcription units including Ad-E7-ΔTmCD40L, Ad-E7-wtTmCD40L Ad-ΔTmCD40L, Ad-wtTmCD40L, and Ad-GFP-ΔTmCD40L were constructed (see materials and methods) (GFP refers to green fluorescent protein).
An in vitro cell free transcription/translation system was used to confirm that the adenoviral vector containing transcription unit encodes the correct molecular weight protein. The results summarized in Table I show that the proteins produced by the in vitro transcription/translation reactions using PCR-generated cDNAs as templates migrated as expected with the following molecular weights: the E7-ΔCtΔTmCD40L is 32 kDa, E7-wtCD40L is 39 kDa, and the ΔCtΔTmCD40L is 22 kDa.
Western blot analysis was used to evaluate the proteins expressed in cells infected with the different adenoviral vectors. The results summarized in Table I show that molecular weights of the proteins produced in the vector infected cells from the Ad-E7-ΔCtΔTmCD40L vector, the Ad-wtTmCD40L vector, the Ad-GFP-ΔCtΔTmCD40L vector, the Ad-wtTmCD40L, and the Ad-ΔCtΔTmCD40L vectors are respectively. 32 kDa, 35 kDa, 50 kDa, 39 kDa and 22 kDa These data confirm that the transcription units are structurally correct and that the vector DNA in each case can be transcribed and translated into protein at sufficient levels to be detected in a Western blot.
The various adenoviral vectors were compared for efficiency of activation of DC cells. For this purpose, bone marrow derived DCs were exposed in vitro to each of the vectors, under conditions that result in infection of close to 100% of the test cells (293 cells). After 48 hours, the cells were reacted with antibodies to activation markers CD80 and CD54 and evaluated in a fluorescent activated cell sorter. The percentage of cells positive for the CD80 and CD54 activation markers on the DCs exposed to the vectors carrying the secretable CD40L transcription units (either the E7/CD40L or the CD40L) was far above that seen with the DCs exposed to the vectors carrying either of the non-secretable CD40L transcription units (wtTmCD40L, or E7/wtTMCD40L), or the Ad-GFP vector as shown below in Table II.
Activation of DCs as measured by the release of cytokines IL-12 and IFN-gamma was determined for the various adenoviral vector transcription units. A statistically significant difference in the level of induction of IL-12 production following exposure of the DCs to the Ad-E7-ΔCtΔTmCD40L and Ad-E7-CD40L vectors was observed (P<0.0001). Specifically, 18�4 pg/2�105 cell/ml/24 h and 88�29 pg/2�105 cell/ml/48 h of IL-12 were produced by the DCs exposed to the AdE7-ΔCtΔTmCD40L vector, whereas exposure of DCs to the Ad-E7-CD40L vector, produced 0 pg/2�105 cell/ml/24 h and 7 pg/2�105 cell/ml/48 h of IL-12.
Similarly, there was a statistically significant difference in the amount of IFN-γ released by DCs exposed to the Ad-E7-ΔCtΔTmCD40L vector (335�29 pg) as compared to DCs exposed to the Ad-CD40L vector (186�9 pg) in first 24 hours (P<0.0001). However, there was no significant difference between the level of IL-12 produced by DCs exposed to the vectors containing the secretable vs the non-secretable E7/CD40L transcription units at 48 hours.
These data indicate that the Ad-E7-ΔCtΔTmCD40L vector is more efficient at inducing a cytokine release from the DCs than the Ad-E7-CD40L vector or the other control vectors. In addition, since the secreted tumor antigen/CD40 ligand complex should engage the CD40 receptor on dendritic cells to effect cytokine release, these results suggest that the E7-ΔCtΔTmCD40L fusion protein forms a functional trimeric molecule at its carboxyl-terminal end to engage the CD40 receptor.
To assess the efficacy of the Ad-E7-ΔCtΔTmCD40L vector for prevention of engraftment of the E7 positive TC-1 cell line in C57BL/6 mice, 1�108 pfu of each vector was injected subcutaneously into mice. Seven days later, a repeat (boost) dose of the same vector was given. One week after the last vaccination, 5�105 TC-1 cells were injected subcutaneously on the back of the C57BL/6 mice at a site separate from the which vector injection site. Following Ad-E7-ΔCtΔTmCD40L vector injection, all of the mice were tumor free at day 108 after tumor challenge. In contrast, following injection with Ad-ΔCtΔTmCD40L; Ad-CD40L; Ad-E7-CD40L, all (5/5) mice developed tumors within 15 days after tumor challenge, and all had died by day 42.
To characterize the types of effector cells involved in the suppression of tumor growth, mice were depleted of CD4 or CD8 T cells by antibody treatment prior to vaccination with Ad-E7-ΔCtΔTmCD40L. Antibody treatment continued even after the TC-1 challenge to ensure proper depletion of the T cells subsets. It was observed that a TC-1 tumor grew in the mice that were depleted of either the CD4 or CD8 T cells. This suggests that presence of both CD4 and CD8 T cells are important for the antitumor effect induced by Ad-E7-ΔCtΔTmCD40L.
To test whether the secretable E7-ΔCtΔTmCD40L protein binds to DCs in the areas close to the site of vector injection, as predicted in the model in
Following the binding of the CD40 ligand to the CD40 receptor, internalization of the tumor antigen/CD40 ligand-CD40 receptor complex and the subsequent digestion and processing of tumor antigen peptides, the DCs migrate to secondary lymphoid organs-regional lymph nodes to become mature DCs. In that location, the activated/antigen loaded DCs can present the tumor associated antigenic peptides and stimulate naive CD4 helper T cells and amplify CD8 cytotoxic T cell lymphocytes (CTL) which are selectively toxic for cancer cells which carry the tumor associated antigen.
In order to test if the injection of AdE7-ΔCtΔTmCD40L vectors induced such a migration of the DCs to the regional lymph nodes in vivo, DCs that were derived in vitro from bone marrow cells were labeled by the supravital dye CFSA and then exposed to vector Ad-E7-CtΔTmCD40L, Ad-E7-CD40L, Ad-ΔCtΔTmCD40L and Ad-CD40L under conditions (100 MOI) designed to generate 100% infectivity. The infected dye loaded DCs were injected into the left flank of the C57BL/6 mice. Three days after these injections, the mice were sacrificed and the axillary lymph nodes on either side were harvested and studied for the presence of the dye loaded DCs. CFDA SE stained DC's carrying the secretable E7/CD40L transcription unit were observed in the regional lymph nodes, whereas CFDA SE stained DC's carrying the E7/CD40L transcription unit were not observed in regional lymph nodes.
CD8+ T cell lymphocytes are involved in protective immune responses to infected or tumor cells. It was examined whether E7-specific CD8+ T cells were induced by subcutaneous injection of C57BL/6 mice with the Ad-E7-ΔCtΔTmCD40L vector. CD8+ T cell responses were assessed using cytotoxicity assays and the detection of antigen-specific CTLs using the enzyme-linked immuno spot (ELISPOT) assay.
Mice were given two subcutaneous injections with the following vectors: (1) Ad-E7-ΔCtΔTmCD40L; (2) Ad-CD40L; (3) Ad-E7-CD40L; (4) Ad-ΔCtΔTmCD40L; and (5) negative control. Spleen cells from vector injected mice were stimulated by exposure to the TC-1 cells at a ratio of 25 TC-1 cells to one spleen cell for 48 hours. The restimulated spleen cells were then plated in 96 cell nitrocellulose filter plates which had been coated with antibodies to either interferon gamma or IL-4. Following the washing of the wells with saline, the ELISPOT assay was carried out. The results of the ELISPOT indicated that the subcutaneous injection of the vector carrying the secretable E7/CD40L protein significantly increased MHC class I-restricted CTLs that were detectable with IFN-γ in spleens to a degree which was greater than that seen following injection of the vector carrying the non-secretable E7/CD40L transcription unit or other control vectors. Also, the frequency of cells producing the T1-type cytokine IFN-γ (117�10.61) was significantly higher than frequency of splenocytes capable of secreting a Th2 cytokine such as IL-4 (22.3�3.68). These data indicate that the E7-ΔCtΔTmCD40L vector vaccination stimulates a Th1 rather than a Th2 immune response.
To determine if CD8 T cell lymphocyte effector cells specifically toxic for cancer cells bearing the E7 antigen were generated following injection of C57BL/6 mice following injection of the vector carrying the secretable E7/CD40L vector, splenocytes obtained animals given two subcutaneous injections of vector were incubated in vitro with mitomycin C-treated TC-1 cells for 5 days for restimulation. Restimulated effector cells were mixed with TC-1 or control target cells for 4 hours and the release of LDH was measured. It was observed that spleen cells from mice injected with the Ad-E7-ΔTmCD40L vector lysed 90% of the TC-1 target cells. No apparent lysis was observed against irrelevant but syngeneic EL-4 cells, which do not carry the E7 antigen. The level of cytotoxicity to TC-1 cells was higher with splenocytes taken from the Ad-E7-ΔCtΔTmCD40L vector inject mice than with splenocytes taken from mice previously injected with the Ad-E7-CD40L vector.
a) Mice
Six- to 8-wk-old C57BL/6 mice were purchased from Harlan.
b) Cell Lines
The C57BL/6 syngeneic TC-1 tumor was immortalized with the HPV-16 E6 and E7 genes and transformed with the c-Ha-ras oncogene (25). TC-1 expresses low levels of E6 and E7 and is highly tumorigenic. TC-1 was grown in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM nonessential amino acids, 1 mM sodium pyruvate, 50 μM 2-ME, at 37� with 10% CO2.
c) Production of Recombinant Adenoviruses
A transcription unit that included DNA encoding the signal peptide from the HGH gene upstream of DNA encoding the full length HPV type 16 E7 protein upstream of ΔCtΔTmCD40L was generated. DNA encoding the human growth hormone signal sequence MATGSRTSLLLAFGLLCLPWLQEGSA (single letter amino acid code) (SEQ ID NO: 1) was prepared by annealing phosphorylated oligonucleotides (SEQ ID NOs: 2 and 3) to generate the full 26 amino acid HGH sequence with Bgl II and NotI overhangs.
Growth hormone signal upper strand (coding sequence in italics):
Growth hormone signal lower strand:
Synthetic HGH signal sequence was prepared by annealing the above upper and lower strand oligos. The oligos were dissolved in 50 μl H2O (about 3 mg/ml). 1 μl from each oligo (upper and lower stand) was added to 48 μl annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM Mg-acetate) incubated at 4 minutes at 95� C., 10 minutes at 70� C. and slowly cooled to about 4� C. The annealed DNA was phosphorylated using T4 PNK (polynucleotide kinase) under standard conditions.
The HGH signal sequence with Bgl II and Not I overhangs was inserted via Bgl II and Not I into pShuttle-E7-ΔCtΔTmCD40L (no signal sequence) to yield pshuttle-HGH/E7-ΔCtΔTmCD40L.
pShuttle-E7-ΔCtΔTmCD40L (no signal sequence) was prepared as follows: Plasmid pDC406-mCD40L was purchased from the American Type Culture Collection. A pair of PCR primers (SEQ ID NOs: 4 and 5) was designed to amplify the mouse CD40 ligand from position 52 to 260 (i.e., without the cytoplasmic and transmembrane domains) and include sequence encoding a linker (indicated as "+spacer") at the 5′ end of the amplicon.
Mouse ΔCtΔTmCD40L+Spacer Forward Primer (MCD40LSPF) (CD40L sequence italicized):
The forward primer MCD40LSPF encoded a 10 residue spacer (FENDAQAPKS; single letter code; SEQ ID NO: 6) to be located between the tumor antigen and the CD40 ligand (mCD40L) of the transcription unit. PCR performed using the forward and reverse primers (SEQ ID NOs 4 and 5) and Plasmid pDC406-mCD40L as the template resulted in PCR fragment "space+ΔCtΔTMCD40L", which was inserted into the plasmid pShuttle-CMV (13) after restriction endonuclease digestion with Hind III (AAGCTT) and Xho I (CTCGAG). This vector is designated pShuttleΔCtΔTmCD40L. A vector was produced that was otherwise the same except that it encoded full length CD40L rather than the truncated form. This vector was made using a CD40 forward primer that annealed to the starting codons of murine CD40L This vector is designated pShuttleCD40L (no signal sequence).
Modification of pShuttleΔCtΔTmCD40L (no signal sequence) to include the HPV-16 E7 upstream of the CD40 ligand sequence was accomplished as follows: Sequence encoding the full HPV-16 E7 protein was obtained by PCR amplifying from the HPV viral genome using the following primers:
A similar procedure as described above was used to insert the E7 encoding DNA upstream of full length CD40 in pShuttleCD40L (no signal sequence).
In a similar fashion, the green fluorescent protein (GFP) gene was inserted downstream of the CMV promoter and upstream the mΔCtΔTmCD40L or mCD40L (wildtype CD40L) and in a replication incompetent adenoviral vector. These vectors were used as controls. Other adenoviral vectors were created by inserting DNA encoding the HGH signal sequence between the CMV promoter and 5′ to DNA encoding ΔCtΔTmCD40L or CD40L of vector pShuttle-ΔCtΔTmCD40L or pShuttleCD40L, respectively, to create vectors pShuttle-HGHΔCtΔTmCD40L and pShuttle-HGHCD40L, respectively.
A transcription unit that included DNA encoding the signal sequence of the mouse IgG kappa chain gene upstream of DNA encoding the full length HPV type 16 E7 protein ("K/E7") was generated by PCR using HPV16 plasmid and the following primers:
A DNA fragment that contained the mouse CD40 ligand from which the transmembrane and cytoplasmic domain had been deleted (ΔCtΔTmCD40L) was generated from a mouse CD40 ligand cDNA Plasmid (pDC406-mCD40L; ATCC) using the following PCR primers:
Adenoviral vector encoding a fusion protein with E7 upstream of full length mouse CD40L was made using primers to amplify full length mouse CD40L. The following primers were used:
The recombinant adenoviral vectors were generated using the Adasy vector system (Strategene, San Diego, Calif.). Briefly the resulting plasmid pShuttle-HGH/E7-ΔCtΔTmCD40L, pShuttle-HGH/CD40L, pShuttle-HGH/E7-CD40L, pShuttle-HGH/GFP, pShuttleHGH/GFP-CD40L, and pShuttle-HGH/E7 were linearized with Pme I and co-transformed into E. coli strain BJ5183 together with pAdEasy-1, the viral DNA plasmid. Recombinants were selected with kanamycin and screened by restriction enzyme analysis. The recombinant adenoviral construct was then cleaved with Pac I to expose its Inverted Terminal Repeats ITR) and transfected into 293A cells to produce viral particles. The titer of recombinant adenovirus was decided by Tissue culture Infectious Dose (TCID50) method.
Primers for amplifying human ΔCtΔTMCD40L+ spacer using a human CD40 ligand cDNA template are set forth below.
Human ΔCtΔTmCD40L+ Spacer Forward Primer (HCD40LSPF) (CD40L sequence italicized):
d) In Vitro Expression of Adenoviral Vector Transcription Units
The coupled in vitro transcription-translation system of RILL (TNT kits from Protege Corp.) was used for the synthesis of the following vectors: Ad-E7-CtΔTmCD40L, Ad-E7-wtTmCD40L, Ad-ΔCtΔTmCD40L, Ad-wtTmCD40L, and Ad-E7. Two μg of each plasmid DNA or PCR generated template DNA was added to the 50 μl reaction mixture containing 25 μl of RRL, 2 μL of TNT reaction buffer, 1 μl of T7 RNA polymerase, 1 μl of amino acid mixture (1 mM) without methionine, 4 μl of [35S]-methionine, and 1 μl of ribonuclease inhibitor RNAsin (40 U/μl). The reaction was completed by incubation at 30� C. for 1 hr. 15 ul of each [35S]-labeled product was run on a 10% SDS-PAGE gel and exposed to X-film overnight.
e) Western Blot Analysis
The cell lysate derived from 293 cells infected by an adenoviral vector at MOI 40 was fractionated on a 10% reducing SDS-PAGE gel and transferred to an IMMOBILON-P membrane (Millipore, Bedford, Mass.). After blocking with 5% nonfat milk for 2 h at room temperature, the membrane was probed with an antibody against the specific mouse CD40 ligand (mCD40L) in TBS-T buffer (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, and 0.5% Tween 20) in the presence of 2% BSA overnight. After washing four times with TBS-T buffer, the blot was incubated with goat anti-hamster alkaline phosphatase conjugated antibody (Jackson Immunoresearch) for 1 hour. The immunoreactive bands were visualized on membrane by using the ProtoBlot II AP system (Promega Corp.).
f) Flow Cytometry Analyses of DC
To quantify the expression level of surface molecules of APCs or DCs, the test cells were stained with FITC- or PE-conjugated anti-mouse monoclonal antibodies (mAbs) for CD80, CD54, and CD11c (Pharmingen), for 30 min on ice, prior to immunostaining with labeled Abs. The APCs were first incubated with a Fc-γ blocking antibody (anti-mouse CD16/CD32 antibody) to avoid the nonspecific binding of mAbs to Fc-γ receptors. The cells were then washed twice, fixed in 4% paraformaldehyde, and analyzed using a Becton Dickinson flow cytometer (FACS Calipur).
g) Cytokine Production of Ad-mCD40-Modified DCs
The DCs were infected with the following adenoviral vectors: Ad E7-ΔCtΔTmCD40L, AdmCD40L, AdE7-CD40L, AdGFP, AdGFP-mCD40L, AdE7 or phosphate-buffered saline (PBS) at MOI 100, and plated in 24-well plates at 2�105 cells/ml. After incubation for 24 hours at 37� C., the supernatant fluid (1 ml) was harvested and centrifuged to remove debris. The level of murine IL-12 or IFN-gamma released into the culture medium was assessed by enzyme-linked immunosorbent assay ELISA), using the mouse IL-12 p70 or IFN-gamma R & D Systems respectively.
h) DC Migration Assays
The protocol used to study the in vitro migration of DCs was the same as that used by Romani et al (21). In brief, the skin of the ear of mice was split in dorsal and ventral halves and dorsal halves were cultured on the bottom membrane of the inner chamber of six well double-chamber tissue culture plate (transwell no 3414, Costar). The outer chamber was loaded with 1�108 vector particles. It was then filled with culture medium so that the vector particles could bath the skin from underneath. The epidermal part of the skin was thus exposed to air. The cultures were incubated at 37� C. for 3 days. The number of DCs that had emigrated from the skin into culture medium was counted under the hemocytometer.
The DC in vivo migration experiment was carried out as follows. After the bone marrow derived DCs were cultured 6 days with IL-3, GM-CSF, they were loaded with the CFDA SE suprevital dye. Briefly, the DCs were incubated with 10 ul of CFDA SE for 15 min at 37� C. The dendritic cells were then pelleted and resuspended in fresh prewarmed medium for 30 min. The cells were mixed with each recombinant adenoviral vector at MOI 200. The vector was then injected into the left flank of the test mouse. Three days later, the axillary lymph nodes draining the region of the injection site for the DCs were removed, frozen tissue sections made and observed in the immunofluorescence microscope.
i) Immunohistochemical Staining
Vaccinated mice were sacrificed 3 days after the Ad E7-ΔCtΔTmCD40L, Ad-ΔCtΔTmCD40L, AdE7-CD40L, AdE7-CD40L vector vaccination. The skin at the site of vector subcutaneous injection was biopsied, embedded in Oct solution, and cut into 5-.mu.m sections. The slides were incubated with rat anti-MHC-II antibody, rat anti-CD40L antibody, and rat anti-CD80 polyclonal Ab (Bioscience). This was followed by exposure to biotinylated goat anti-rat IgG antibody (1:200 dilution) and avidin-biotin complex (1:100 dilution; Vector, Burlingame, Calif.). The stained slides then were mounted, and studied under a fluorescence microscope.
j) Cytokine Profile by ELISPOT Assays
The presence of E7-specific effector T cells in the immunized mice was also assessed in ELISPOT assays, as previously described (26, 27). Briefly, splenocytes obtained from mice vaccinated with each of the different vectors were restimulated in vitro by culture with the TC-1 cell line (responder-to-stimulator ratio 25:1) in the presence of 10 U/ml IL-2 for 48 hours. Re-stimulated splenocytes were then plated in 96-well nitrocellulose filter plates (5�104 cells in 100 microliters). The wells were precoated with rat anti-mouse anti-IFN-antibody or anti-IL-4 antibody. After incubation for 24 hours at 37� C./5% CO2, the plates were then washed with PBS, and the presence of cytokine-producing spleen cells was detected by incubation at 4� C. with biotinylated goat anti-rat secondary antibody, followed by 100 microliter/well horseradish peroxidase avidinD and 150 microliter/well freshly prepared substrate buffer (0.4 mg/ml 3-amino-9-ethyl-carbazole in a total of 50 ml 0.05 mol/L sodium acetate buffer) and 20 microliter 30% H2O2. The stained spots corresponding with IFN producing cells or IL-4 producing cells were enumerated under a dissecting microscope.
k) Cytotoxicity Assay
For these studies, mice received vaccination via a subcutaneous injection of the following vectors: Ad-E7-ΔCtΔTmCD40L (secretable CD40 ligand), Ad-ΔCtΔTmCD40L (secretable CD40 ligand), Ad-E7-CD40L (non-secretable CD40L), Ad-CD40L (non-secretable ligand) and Ad-E7. The vectors were injected on days 0 and 7 as a subcutaneous injection. As a control, PBS was injected subcutaneously on days 0 and 7 (control mice). Mononuclear cells from the spleens of these mice (vector injected or PBS injected) were used as the source of effector cells for cytotoxicity studies. Aliquots of the spleen cell suspensions were then co-incubated with mitomycin C-treated TC-1 cells in RPMI 1640 medium, supplemented with 10% FBS, 50 M 2-mercaptoethanol, 2 mM glutamine, 1 mM pyruvate, and nonessential amino acids, under cell culture conditions for 5 days. To perform the cytotoxicity assay, 5.times.10.sup.3 of TC-1 tumor cells (target cells) were incubated with the stimulated splenic mononuclear cells (effector cells) at an effector/target ratio of 100:1 for 4 hours at 37� C., in culture media containing 5% FBS. At the end of the incubation, mononuclear cell-mediated cytotoxicity was determined using the nonradioactive cytotoxicity assay kit. Released LDH in culture supernatants was measured by an ELISA plate reader. The data were calculated as: % CYTOTOXICITY=(EXR ESR TSR+CMB/TMR TSR VC+CMB) 100 where EXR is experimental LDH release, ESR is effector cell spontaneous LDH release, TSR is target cell spontaneous LDH release, TMR is target cell maximum LDH release, VC is volume correction, and CMB is culture medium LDH back-ground. Statistics: Student's unpaired t-test was used to determine the differences between the various groups in the proliferation and cytotoxicity assays. Statistical significance was determined at the 0.01 level.
l) In Vivo Efficacy Experiment in Mouse Model
Mice (5 or 10 per group) were vaccinated via subcutaneous injection with 1�108 PFU Ad-E7-ΔCtΔTmCD40L, Ad-E7, Ad-E7-CD40L, Ad-ΔCtΔTmCD40L or Ad-CD40L vectors. One week later, mice were boosted with the same adenoviral vector regimen as the first vaccination. One week after the last vaccination, mice were to challenged by subcutaneous injection of 5�105 TC-1 cells/mouse in the right leg and then monitored twice a week.
The donor splenocytes were harvested from tumor-free donor C57BL/6 mice and passed over magnetic columns to enrich for total T cells. Tumor growth was measured as described previously.
m) Statistics
All parameters were analyzed using Students t test, or ANOVA followed by Scheffe's procedure for multiple comparisons as post-hoc analysis; all data shown is presented as mean�S.E. of the mean (S.E.).
All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the a and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
Other embodiments are set forth within the following claims.
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text:
Ginger Cocktail
9 Oct 2010
|Lemon juice
|2 Cup (32 tbs)
|500 Gram
|4 Cup (64 tbs)
|6 Cup (96 tbs)
|Potassium meta bisulphite
|2 1⁄2 Gram
Peel ginger and cut into pieces.
Add 1/4 cup water and liquidise to crush ginger.
Strain the juice.
In the residue, add 1 1/4 cup water and liquidise again.
Strain the juice.
Mix both the extractions.
Combine sugar and 3 1/2 cups water in a pan and place on fire.
Stir till sugar is dissolved.
When it comes to a boil, add a little lemon juice to clarify the syrup.
Let boil for a minute more.
Remove from heat.
Add ginger juice and strain the syrup.
When thoroughly cold add lemon juice.
Dissolve preservative in four tablespoons water and add to the cocktail.
Fill the cocktail in sterilized bottles leaving a gap of 1 1/2 inches on the top.
Cork and seal the bottles.
Store bottles in a cool, dry place.
Recipe Summary
Course:Wine And Drink
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Ginger Cocktail Recipe

subset: commoncrawl


text:
affect children's health. Data systems can also be used to communicate knowledge about children's health to the public. Integrated data systems provide early information to identify areas of vulnerability, monitor health disparities, and detect manifestations of adverse effects on children's health across time, across domains of health, or for a variety of subpopulations defined by geography, ethnicity, or other characteristics.
Integrated data systems with data linked at the individual level also allow individual children or groups of children to be followed longitudinally across datasets to better understand why a characteristic or problem develops in some children but not others, and they support epidemiological studies of the potential effects of certain health influences. Linked datasets from multiple service systems, such as public health, education, human services, child welfare, juvenile justice, and health care, enable particularly useful longitudinal analyses. Based on guidelines to protect privacy and confidentiality, there should be limited access to linked data systems for a range of purposes. For example, policy makers can forecast and monitor the effect of their policies on children; researchers can determine best practices for health, social, and education services; the particular impact of multiple health influences on children can be determined; and management of patient care can be improved.
A community can be empowered when it has information specific to its children. States and some communities collect a wealth of data about children and their health. If data at the state or community level are scattered among several agencies or departments, it is difficult for community groups to compile them. Community groups and local policy makers are much more likely to use data systems that combine administrative datasets from multiple state and community sources (without identifiers) and are easily accessible to the public. Making children's data systems accessible on the Internet can enhance distribution and use by individuals outside government and the research community. The responsibility for providing much of these data is the public health department at the state and local levels. Since one of the three core functions of public health is assessment, data collection and dissemination is a major activity. The Institute of Medicine recommended in 1996 that the state health department be responsible for the provision of data. More specifically, within the public health department, the Title V maternal and child health program is mandated by federal law to be the lead on maternal and child health issues.
Advances in the systematic collection, analysis, and reporting of data have been made in recent years. Standardizing data definitions and collection methods is an important step in improving the value of data elements, datasets, and data systems and has been a priority of the National Center for Health Statistics (NCHS). Federal, state, and community programs have been encouraged to adhere to guidelines established by NCHS in order to improve the comparability of data. Without standardization, even sophisticated, integrated data systems cannot provide good information.


text:
Aztec Camera's cover of Van Halen's "Jump" is possibly my favorite cover of all time. It was the b-side to the 12 inch version of their single "Oblivious." I listened to "Oblivious" once, flipped the record over and then listened to this for about four hours straight.
For sheer "What the hell" reaction, here's Gwar covering Kansas's "Carry on" courtesy of AV Club's awesome AV Undercover series.
And a bonus: Yo La Tengo doing the New York Mets theme song, "Meet the Mets."


text:
l SQA Candidate Advice Line
SQA's Candidate Advice Line will be available until Friday 8 August. Candidates can ring 0345 213 1000 for advice on the content and layout of their certificate.
l UCAS website
All the information students need to access clearing is on the UCAS website www.ucas.com and expert advisers are also on hand to answer questions on 0371 468 0 468. Their social media team are also helping applicants on Twitter and Facebook.
l SDS Results Helpline
Skills Development Scotland is offering a helpline for candidates who have questions about their next move on 0808 100 8000.


text:
Tel Aviv Travel Guide
Getting Oriented
Tel Aviv's compact size and flat landscape make it easy to get around on foot. The city's main north–south thoroughfares of Hayarkon, Ben Yehuda (which becomes Allenby), Dizengoff, and Ibn Gvirol streets run more or less parallel to the Mediterranean shoreline. Closest to the water is Hayarkon and the beachfront Tayelet (promenade). At the northern end of Hayarkon is the Tel Aviv Port. Most hotels are on the beachfront along Hayarkon.


text:
Respond to Review of YuShanGe
by camerongreen
Review: This is one of the best vegetarian restaurants in the world. The presentation is just so lovely, they bought me a tiny sesame dumpling in a jar, on a piece of driftwood, with an orchid in a vase on it. I didn't want to eat it, but when I did, that dumpling was one of the most beautiful things I've ever put in my mouth. This was just one of 10 courses. I went back on my last day in Taipei (and to Ever Green buffet) and even though frankly I was dressed like a hobo by Taiwanese standards, the waitresses were all really happy to see me again and treated me even better than the first time. It's expensive, but you only live once, so pay whatever they ask, think only about how beautiful food can look and taste and how lucky you are to be there, and savour the moment as it might be a long time before you get a chance to eat at a place this good again. OK having said all that, as a vegan they did serve me a drink with honey in it, so make sure you let them know when you order.
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31 year old man | Springfield, VA, USA | Seeking women 25-35 within 50 miles
Love from Asia looking for a " I and Ana" scene (coming from "King and I" movie). I
Originally from China, I've been living in the U.S. for almost 10 years. It is hard to conclude myself in one sentence, but I can tell you that I am not an "average Joe" type of person though I may look like... (guess all Asian look the same when they wear glasses.) I have a good sense of humor. I like to make people laugh . Because of my often hectic schedule, I feel that a good sense of humor is the best way to relieve stress and lighten the spirit. I am certainly looking for the one who I can relieve and lighten her. I think the most beautiful thing will be the smile I can bring to her. I have a strong awareness of healthy-style life. I like to do workouts in gym, running on the streets and play ball game with my friends. I want her to have the same interest . It not only benefits health but relationship as well. I always dream me and my love running , biking , hiking and working out together. I have high recognition of multi-culture integration. I'd like her coming from a different culture background but willing to learn another culture and mix two different world together. I firmly believe no mater what culture she comes from, what religion she believes and what nationality she belongs, love is always there. I'm looking for a companion–somone who is independent, with a strong personality, yet also able to make the compromises that life sometimes requires. Someone who has an athletic body and a healthy life style with a willingness to learn a different culture . She shall be able to engage different activities and laugh at herself. If you are interested in me, please wink at me. I will be there for you !
Camping , Coffee and conversation , Business networking , Cooking , Dining out , Movies/Videos , Music and concerts , Nightclubs/Dancing , Playing sports , Religion/Spiritual , Travel/Sightseeing , Volunteering , Watching sports
Running, Swimming, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I like Birds, Dogs, Fish
Middle of the Road
Ultra Bar. Eden Club, Cafe Asia, Mansion on O St, Ibiza
Movies, Eating out. Going to a adventure . Extreme Sports, Hiking and Camping together
Outdoor sports and sightseeing including city touring, museums touring and historic sites visiting. Traveling will be another.
A Day in My Dream !
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'11" (180cms)
|4'5" (134cms) to 5'6" (167cms)
|Body type:
|Athletic and toned
|Big and beautiful, About average, Athletic and toned
|I'll tell you later
|No preference
|No preference
|No Way
|No Way
|Social Drinker
|Social Drinker
|Financial / Accounting / Real Estate
|Administrative / Secretarial, Artistic / Creative / Performance, Financial / Accounting / Real Estate, Medical / Dental / Veterinary / Fitness, Political / Govt / Civil Service / Military, Sales / Marketing, Self-Employed / Entrepreneur, Education / Teacher / Professor, Nonprofit / Volunteer / Activist, Fashion / Model / Beauty, Architecture / Interior design
|$50,001 to $75,000
|$35,001 to $50,000, $50,001 to $75,000
|Never Married
|Never Married
|Have kids:
|Wants kids:
|Definitely, Someday, Not sure, Probably not
|Asian, Black / African descent, East Indian, Middle Eastern
|Christian / Protestant
|Christian / Protestant
|Chinese, English
|Chinese, English
|Graduate degree
|Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


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Hollywood star Nicole Kidman was presented with Australia's top civil honour earlier today (13Apr07) for her work on the big screen, as well as her efforts to promote women's health.
The Oscar-winning Moulin Rouge! star was awarded the Companion of the Order of Australia at Government House in Canberra, the country's highest honour, which is given to citizens for their 'eminent achievement and merit of the highest degree in service to Australia or humanity at large.'
Kidman was presented the honour - the equivalent to an English knighthood - in her homeland by Governor-General Michael Jeffrey, who praised her promotion of women and children's health and cancer research.
She gushes, "It's a pat on the back, which as a little girl is something you dream of."


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Two blocks, each of mass 1.5 kg, are attached to links which are pin-connected to bar BC. as shown. The masses of the links and bar are negligible, and the blocks can slide without friction. Block D is attached to a spring of constant k = 720 N/m. Knowing that block A is at rest when it is struck horizontally with a mallet and given an initial velocity of 250 mm/s, determine the magnitude of the maximum displacement of block D during the resulting motion.
Fig. P19.70


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Lagos — The Federal Government has released about N1 billion premium for the group life policy for workers this year. The N1 billion is about 25 percent of the sum premium for the staff covered under the policy.
This could be a fallout from the visit to the Head of Service by Nigerian Council of Registered Insurance Brokers (NCRIB).
The Head of the Civil Service of the Federation, Alhaji Isa Bello Sali, had directed that all outstanding premiums for the 2012 Federal Government Group Life Assurance Scheme should be paid without further delay.
He gave the directive in his office in Abuja when he received a delegation of the Governing Board of the Nigerian Council of Registered Insurance Brokers led by its President, Barrister Laide Osijo.
The Head of Service who was represented by the Permanent Secretary, Common Services Office, Alhaji Bukar Goni Aji, acknowledged the important role of insurance brokers in the successful implementation of the Group Life Assurance Scheme and also assured them that as a body established by law, the council will continue to receive appropriate prominence in all issues related to the scheme.
Speaking further, he urged the council to ensure that all its members operate in line with laid down rules and ensure that beneficiaries of the scheme receive prompt payment and are well treated.
Earlier in her address, the President of the Nigerian Council of Registered Insurance Brokers, Barrister Laide Osijo, called on the Office of the Head of the Civil Service of the Federation to reverse the order of issuance of appointment letters for participation in the scheme.
According to her, a situation where underwriters receive letters of appointments before brokers may limit the effectiveness of brokerage services and is not in line with international best practices.
Barr. Osijo also stressed the need for government to pay greater attention to the insurance of its non-life assets in line with laid down rules. If this is adhered to, she added, the insurance industry will be more robust in its contribution to national economy.


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Barrie-Muskoka > Recreation
FoundLocally is committed to providing one-stop information to people about
Barrie & the Muskokas. Here are is key recreation and entertainment information for Barrie, Orillia, the Georgian Bay shore, the Bruce Peninsula, and the Muskoka Lakes:
Below the recent postings are our Barrie-Muskoka Recreation & Entertainment.
No Arts & Entertainment Events in the next two weeks have been submitted.
No News Announcements have been posted in the past 2 weeks.
No items have been added/updated in the past 2 weeks.
If it's movies you want, it's movies we give! We have movie showtimes, updated by Thursday noon, to help plan your weekend. We have nice capsule movie reviews to decide which flicks are worthwhile, and even "Top 15" movie rankings each Friday showing what theatre owners think of the movies (why wait for Monday's weekend movie grosses?).
Checkout the other things happening in the local entertainment scene, as well as the many festivals throughout the year.
Barrie & the Muskokas has over 200 restaurants, ranging from diners and fast food for families and a quick bite, up to the four-star establishments where a nice dinner for two can easily be a hundred dollars, before wine. Barrie and the Muskoka Region is more than steaks, and has its fair share of seafood and sushi, as well a full complement of almost any ethic cuisine you can imagine. The city's youth also make for thriving nightlife.
Check out the area's bars, lounges, pubs and coffee shops. There's lots of places to go out and meet your friends (or ones you don't know yet!)
There are lots of sports opportunities in Barrie & the Muskokas. Yes, we have some semi-pro competitive sports, but Barrie is an active city.
It seems everybody is doing something to stay in shape. FoundLocally has descriptions and information about over 40 sports. Get involved, alone, on a team, or in a league!
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The National Institute of Neurological Disorders and
Stroke (NINDS), a part of the National Institutes of Health, is the leading
U.S. federal government agency supporting research on brain and nervous system
disorders. It provides the public with educational materials and information
about these disorders.
NCBDDD aims to find the cause of and prevent birth
defects and developmental disabilities. This agency works to help people of all
ages with disabilities live to the fullest. The website has information on
many topics, including genetics, autism, ADHD, fetal alcohol spectrum
disorders, diabetes and pregnancy, blood disorders, and hearing loss.
This website is sponsored by the Nemours Foundation. It
has a wide range of information about children's health, from allergies and
diseases to normal growth and development (birth to adolescence). This website
offers separate areas for kids, teens, and parents, each providing
age-appropriate information that the child or parent can understand. You can
sign up to get weekly emails about your area of interest.
The March of Dimes tries to improve the health of babies
by preventing birth defects, premature birth, and early death. March of Dimes
supports research, community services, education, and advocacy to save babies'
lives. The organization's website has information on premature birth, birth
defects, birth defects testing, pregnancy, and prenatal care.
The National Dissemination Center for Children with
Disabilities (NICHCY) is the national information and referral center that
provides information on disabilities and disability-related issues for
families and professionals. The focus is on children and
youth, birth to age 22.
United Cerebral Palsy (UCP) offers family support for people with cerebral palsy. UCP publishes
newsletters, brochures, pamphlets, scientific reports, and books. UCP also
works for the rights of people with cerebral palsy. There are chapters in every
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Finding the best schonbek for your home can be an overwhelming task. By browsing our schonbek categories or by calling one of our trained experts, we can easily help you find the best fixtures for sale.
Manufacturer Information
About Schonbek
Adolf Schonbek started Schonbek in 1870 after leaving his family's glassworks to start his own business. By pairing crystal and light together, he was able to create some of the most extravagant chandeliers ever seen. For over 140 years, Schonbek has remained the world's leading producer of crystal lighting. In 2007, Schonbek became part of the Swarovski Company and, together, they bring over 255 years of tradition, innovation, and quality to the table, resulting in beautiful lighting fixtures.
Where You Can Find Schonbek's Famous Crystals
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Mississippi Decides To Finally Abolish Slavery, Because of Movie
Dr. Ranjan Batra immigrated to the United States from India and became a U.S. citizen four years ago. With the zeal typical of the new citizen, Batra asked somebody to look on a website, which led to the discovery that Mississippi had never ratified the 13th Amendment outlawing slavery. But things move more slowly in the South, so why should an outsider doctor bother the great state of Mississippi just because it ignored the 13th Amendment it was supposed to ratify 148 years ago?
Mississippi's legislature first got around to voting on the 13th Amendment in 1995, with several elected politicians in the southern state actually abstaining from the vote because aren't black people supposed to be slaves? But the state never filed the proper paperwork with the federal government, and it probably would've stayed that way forever if not for Dr. Batra seeing Lincoln and having the obvious thought that probably Mississippi was not really on board with the whole 13th Amendment thing from 1865.
Mississippi is one of nine southern states—including honorary southern states Arizona and Alaska—where the Voting Rights Act of 1965 is in force, because of persistent racism. An overwhelmingly white and Republican county in neighboring Alabama is currently trying to overturn the Voting Rights Act. The Supreme Court will hear this case at the end of February. And in the cold wet ground beneath Lincoln's Tomb, a long-dead hero begins to stir ….


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Freshman Application
Fall 2015 Freshman Application
Welcome to the Fall 2015 Kellogg Honors College freshman application. Applicants must have applied to Cal Poly Pomona as a first-time freshmen by November 30, 2014 to be eligible for the Kellogg Honors College. This application is completely electronic/online. Applicants must log in using their BroncoNumber and Password, which are the same credentials used to access BroncoDirect. The application requires submission of personal academic information, essay question responses, and one (1) high school teacher letter of recommendation. Teachers can submit a letter of recommendation through this webpage. Test scores and GPA information will be downloaded from applicants' Cal Poly Pomona application. The deadline for all application materials will is 5:00PM on February 18, 2015 8:00AM on Thursday, February 19, 2015.
The Kellogg Honors College freshmen application closed on February 19, 2015.
Admissions decisions will be sent via email in the third week of March 2015.
Frequently Asked Questions:
When is the application deadline?
- 5:00PM on Wednesday, February 18, 2015 8:00AM on Thursday, February 19, 2015
What are the minimum requirements for freshmen applicants?
- 3.50 cumulative high school unweighted GPA recommended
- Exemption from EPT and ELM placement tests at Cal Poly Pomona
How can a teacher submit a letter of recommendation?
- Teachers can submit a letter of recommendation at this website. The letter of recommendation must be received by 5:00PM on February 18, 2015 8:00AM on Thursday, February 19, 2015.
What is my username and password to access the application?
- The username (BroncoName) is your Cal Poly Pomona email address without the @cpp.edu. For example: if your email is email@example.com, your username is billybronco.
- The password is the same credential used for BroncoDirect. Please call the Cal Poly Pomona HelpDesk at 909-869-6776 or visit their website for assistance.
Will admission to the Kellogg Honors College increase my chances of admission to Cal Poly Pomona?
- The admissions processes for Cal Poly Pomona and the Kellogg Honors College are separate. Although the Kellogg Honors College's minimum admissions requirements are higher than Cal Poly Pomona's, admission to the Kellogg Honors College does not increase students' admission chances to Cal Poly Pomona.
Can I apply if I am a current Cal Poly Pomona freshmen student?
- Current Cal Poly Pomona students in their first year as first-time freshmen can apply to the Kellogg Honors College through the sophomore application.


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The Columbia City Church of the Nazarene Alpha team won the Church League Softball championship recently. The squad finished the season with a 14-2 record. Pictured, front row, from left: Kyle Shew, Bo Sallee, Marissa Sallee, Carrie Dunn and Andrew Davison. Second row: Clayton Clifford, Travis Janssen, Maddy Minnick and Chuck Minnick. Back row: Josh Smith, Hunter Coyle, John Dunn, Todd Coyle and Jason Shoemaker. League games were played at First Church of God from May thru August.View more articles in:


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Trinity Stores
"Trinity Stores is the type of café-cum-deli that brings the village feel back to a suburban neighbourhood, or, in this case, Balham," statesMatthew. "It has open floor-to-ceiling shelves with all the foodie brands you'd expect and plenty more. There's an excellent selection of salads, tarts and quiches, plus the best scotch eggs and sausage rolls south of the river. Boxes of Amalfi lemons, wild rocket and freshly baked breads greet you as you enter."
Where: 5-6 Balham Station Road, London SW12 (020-8673 3773; www.trinitystores.co.uk)
© independent.co.uk


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Burgundy leather color pouch
Has anyone had any experience using burgundy leather color?
I recently bought the white color leather and its get dirty really fast and lost its original color in a few weeks time. It is frustrating!
If anyone had used the burgundy leather color, how does it look after some time? does it gets dirty and lost its originality after daily used? because I'm thinking to buy the burgundy leather color
- Asked by Bree O from Orlando
Sena Ultraslim Pouch for iPhone 4
Product No Longer Available
No Answers have been submitted


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Oracle aims to dethrone IBM in business hardware
Oracle boss Larry Ellison said that he is out to dethrone IBM in the realm of business network hardware, including high-end computer servers.
Oracle boss Larry Ellison said that he is out to dethrone IBM in the realm of business network hardware, including high-end computer servers.
Apple chief Tim Cook on Tuesday vowed that creativity would remain in the company's "DNA" and hinted that products on the horizon could come in the area of television.
US media giant News Corp. has taken a 19.9 percent stake in Bona Film Group, which calls itself the largest privately owned film distributor in China, the firms said.
(AP) -- If the phone hacking scandal gripping Rupert Murdoch's News Corp. empire has a familiar ring, it might be because you've heard the story before. Scrappy outsider turns modest newspaper business into international ...
The Los Angeles Times said Friday that it will begin charging online readers next month, the latest major US newspaper to require a subscription to its website.
A British newspaper has thrown open its office doors, let the readers stride in, and invited them to peer over reporters' shoulders -- digitally, at least.
Rupert Murdoch's News Corp. posted a slight decline in quarterly net profit on Wednesday, partly due to one-time charges stemming from the phone-hacking scandal in Britain.
Former News Corp. boss Peter Chernin said Friday he will not rule out buying Yahoo! but suggested the time was not right, amid intense speculation on the future of the struggling US Internet giant.
The Australian arm of Rupert Murdoch's News Corporation will begin charging for online content, the firm said on Thursday, with its flagship national broadsheet to move behind a paywall from next week.
The Boston Globe on Monday became the latest major US newspaper to begin charging readers online.
Apple Inc. has pulled the plug on its 99-cent rentals of television show episodes after making a push last year to persuade TV networks to sign on to the plan.
Google chairman Eric Schmidt has attacked the British education system, saying a failure to appreciate the importance of computer science was holding the country back in the digital age.
Rupert Murdoch claimed boardroom backing Wednesday to remain at the helm of News Corp. as he worked to shift attention from the phone-hacking scandal to his media behemoth's healthy bottom line.
Media behemoth News Corp. posted strong profits Wednesday, boosting owner Rupert Murdoch's attempts to reassure investors after a damaging phone-hacking scandal in Britain.
(AP) -- A British newspaper owned by Rupert Murdoch is warning some readers that their personal details may have been stolen during a cyberattack.


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The great "Fabulous Moolah" passed away on Friday.
I remember watching her wrestle at the Grand Olympic in Los Angeles back in 71 or 72. I was a big "Wrasslin Fan" back then and she always gave a great preformance.
My grandmother, who only spoke spanish, was also a big fan. She never missed a "Moolah" match on TV when possible and refered to her as "Un Hombre".


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15 things we can't wait to do at camp
Is he flirting with you or just a friendly dude?
Misty Copeland becomes the first African American woman to lead the American Ballet Theater!
Book Club: Win this *wicked* read (before you watch the movie!)
Love is in the air, Aquarius!
DIY statement earrings for the budget fashionista
This fruit offers some *serious* skin benefits
Why you should be flossing...even if you're too lazy to do it
Have you ever been bullied? Share your story to help other girls!
GL + Disney take a look at the Teen Beach 2 premiere
More Friends = More Fun
GL Tweets !
4 HOURS AGO Future fling or friend zone? Get the scoop on flirty dudes: http://t.co/nTGyYyjthK pic.twitter.com/CpO9UHDZe3
5 HOURS AGO .@lauramarano is seriously the best: http://t.co/GS0v0nkF5l pic.twitter.com/mnrfWBIoUb
6 HOURS AGO Never trust a text that you get at 3am: http://t.co/lVcPwj8pJn pic.twitter.com/tGIVdYfJWQ
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22 Comments | Add Yours
Coraline is awesome! It's a bit weird, but I love it!!!! The book is way creepier.
by Gabriella123 on 10/27/2012 8:04:04 AM
The Woman in Black didn't really scare me. I only liked it because it had Daniel Radcliffe XD
by Gabriella123 on 10/27/2012 8:02:51 AM


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Small shoulder bag in imitation leather. Flap with zip, narrow shoulder strap, and one inner pocket with zip. Lined. Size 5 1/2 x 7 1/2 in.
100% polyurethane.
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Reach tens of thousands of people instantly by advertising with Ekklesia. Find out more
The 'Big Bang' has reopened questions about the creation of the universe.
Does science lead inexorably to atheism?
- Do you volunteer in an existing befriending scheme?
- Would you like to share your experience with others who want - to do something similar?
What is the responsibility of the international community when a state is unable or unwilling to protect its own people?
A talk organised by Quaker Peace & Social Witness and Peace News at Friends House, Euston Road, London, NW1 2BJ 7.30pm.
Come to the Fellowship of Reconciliation's Called to be Peacemakers conference, a residential weekend event for 18 to 30 (ish) year olds who are interested, active and/ or engaged in peace and confl
A Day Exploring Spiritual Traditions and the Environment
Abbot Christopher Jamison (Worth Abbey/The Monastery TV Series)
Speaking on the theme The Ecology of Happiness
The Centre of African Studies (London), based at the School of Oriental and African Studies (SOAS), is pleased to announce a two-day conference on 'Researching Violence and Conflict: Methodological
It's 100% wrong...
A day conference, organised by Peace Brigades International UK, will explore threats to biodiversity and climate change presented by deforestation, pollution and mono-crop cultivation.


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Browse Flip-Lid Cases for iPad 2
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|Seidio EXPERT Case for iPad 2
|Protect and conveniently transport your iPad 2 with the sleek and eco-friendly, EXPERT case.
|$39.95 $54.95
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