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US 6881405 B2
The invention is an apparatus and method for the treatment of prostate cancer, and involves administering an antibody that specifically binds with prostate antigen.
1. A method for inducing an immune response comprising administering to a mammal an antibody or antigen-binding fragment thereof that specifically binds to an epitope of circulating prostate specific antigen, said epitope being the epitope to which a monoclonal antibody produced by a hybridoma that has ATCC Designation Number 1-18-12526 specifically binds.
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This application claims the benefit of U.S. Provisional Application No. 60/089,281 filed on Jun. 15, 1998.
The present invention relates generally to the generation of cellular and humoral immune responses to prostate-specific antigen (PSA). The present invention also relates to a therapeutic composition that contains a monoclonal antibody that is useful in generating cellular and humoral immune responses to PSA.
Prostate cancer is one of the most common cancers in men and is one of the most common causes of cancer death, although, if diagnosed early, is potentially curable through surgical intervention, radiation therapy, or hormonal therapy. Since differential diagnosis is difficult at best, however, most prostate cancer is not diagnosed until later stages, typically after metastasis of the primary tumor. The primary therapy for metastatic prostate cancer is either androgen-antagonistic agents or castration, but most relapse patients show prostate tumor cells that are androgen-independent. Currently, there are no effective chemotherapeutic agents that can control the growth of androgen-independent prostate tumor cells.
PSA is a 240 amino acid member of the glandular kallikrein gene family. This 33 kDa single chain glycoprotein is a serine protease secreted by both normal and transformed epithelial cells of the prostate gland. PSA can be detected at low level in the sera of healthy males without evidence of prostate cancer. However, during neoplastic states, circulating levels of this antigen increase markedly, correlating with the clinical stage of the disease. PSA expression is almost exclusively restricted to the prostate cells, and is the most widely used marker for the diagnosis and monitoring of prostate cancer patients.
The tissue specificity of PSA makes it a particularly attractive target antigen for the development of immunotherapies against prostate cancer. PSA is detected in nearly all adenocarcinomas of the prostate and the expression of PSA was also demonstrated at distant metastatic sites. Several studies point out that PSA is a potential target for the induction of T-cell directed immunity against prostate cancer. Indeed, several PSA peptides capable of binding to MHC-class I molecules have been identified and it is now well established that a PSA specific T-cell repertoire exists in humans. Studies using in vitro immunization have shown the generation of CD4 and CD8 cells specific for PSA and T-cell lines with specific recognition of PSA peptides were generated from both healthy volunteer and prostate cancer patients. Of particular importance is the generation in vitro of cytotoxic T-lymphocytes specific for PSA and capable of lysing human prostate tumor cells [Xue et al., Prostate 30(2):73-8, (1997); Correale et al., Journal of the National Cancer Institute 89(4):293-300 (1997); Correale et al., Journal of Immunology 161(6):3186-94 (1998); Alexander et al., Urology51(1):150-7 (1998)].
Based on these promising data, numbers of PSA-targeted immunotherapeutic approaches are currently being investigated in preclinical setting. Clinical trials employing a recombinant vaccinia virus engineered to express PSA, recombinant PSA encapsulated into liposomes, or autologous dendritic cells loaded with peptide sequences of PSA have already started [See Future Oncology Vol 4, No. 3/4 (1998)].
The present invention describes a new immunotherapeutic product for prostate cancer that employs as a therapeutic agent a binding agent such as a monoclonal antibody directed against PSA. It is believed that the therapeutic efficacy of the injected anti-PSA antibody is based on multiple mechanisms of actions acting in synergy. Tumor cell killing through an ADCC or CDC mechanism is not expected for these binding agents since PSA is not expressed at the cell surface. The therapeutic efficacy of these binding agents relies on the induction of a specific PSA cellular immune response and in the non-specific stimulation of the host immune system resulting in the induction of an immune response against various tumor antigens.
The induction of a specific cellular immune response upon immunization of the host with either Ab1 or Ab2 has been demonstrated in a number of studies. Of particular importance is the generation through this mechanism of specific CTLs responses in ovarian cancer patients, melanoma patients, myeloma patients, and non-Hodgkin's lymphoma patients [Nelson et al., Blood 88(2):580-9 (1996); Madiyalakan et al., Hybridoma 16(1):41-5 (1997); Osterborg et al., Blood 91(7):2459-66 (1998); and Pride et al., Clinical Cancer Research4:2363 (1998)]. It is therefore expected that the immunization of prostate cancer patients with the binding agents of the present invention may also induce a specific and protective CTL immune response against PSA.
This makes PSA an attractive target for immunotherapy. Several attempts at generating an immune response have met with limited success [Xue et al, The Prostate, 30:73-78 (1997); Correlae et al, J. National Cancer Institute, 89:293-300 (1997); Choe, et al, Cancer Investigations, 5:285-291 (1987); Wei, et al, Cancer Immunol. Immunother., 42:362-368 (1996), and International Application No. PCT/US97/04454, filed 19 Mar. 1997]. Combining the profound impact of prostate cancer with the lack of effective therapies, it is clear that alternative modalities of treatment need to be explored and that the ability to elicit a therapeutic immune response to PSA would be highly desirable.
Immunotherapies involve one or more components of the immune system to trigger a complex cascade of biological reactions focused on eliminating a foreign molecule from the host. The immune system consists of a wide range of distinct cell types, the most important of which are the lymphocytes. Lymphocytes determine the specificity of immunity, and it is their response that orchestrates the effector limbs of the immune system. Cells and proteins, such as antibodies, that interact with lymphocytes play critical roles in both the presentation of antigen and in the mediation of immunologic functions.
Individual lymphocytes provide a specialized function by responding to a limited set of structurally related antigens. As noted in more detail below, this function is defined structurally by the presence on the lymphocyte's surface membrane of receptors that are specific for binding sites (determinants or epitopes) on the antigen. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions. One class of lymphocytes, B cells, are precursors of antibody-secreting cells, and function as mediators of the humoral immune response. Another class of lymphocytes, T cells, express important regulatory functions, and are mediators of the cellular immune response.
Cancer immunotherapy is based on the principle of inducing the immune system to recognize and eliminate neoplastic cells. The key elements in any immunotherapy is inducing the host immune system to first recognize a molecule as an unwanted target, and then inducing the system to initiate a response against that molecule. In healthy hosts, the immune system recognizes surface features of a molecule that is not a normal constituent of the host (i.e., is “foreign” to the host). Once the recognition function occurs, the host must then direct a response against that particular foreign molecule.
Both the recognition and the response elements of the immune system involve a highly complex cascade of biological reactions. In most immunologically based disorders, at least one of the steps in the recognition phase, or at least one of the steps in the response phase, are disrupted. Virtually any disruption in either of these complex pathways leads to reduced response or to no response. The inability of the immune system to destroy a growing tumor has been attributed, among other factors, to the presence of tumor-associated antigens (TAA) that induce immunological tolerance and/or immunosuppression. For example, in some kinds of cancer, the cancer itself tricks the host into recognizing a foreign cancer cell as a normal constituent, thus disrupting the recognition phase of the immune system. The immunological approach to cancer therapy involves modification of the host-tumor relationship so that the immune system is induced or amplifies its response to the TAAs. If successful, inducing or amplifying the immune system can lead to tumor regression, tumor rejection, and occasionally, to tumor cure.
One of the host system's mechanisms for combating a foreign molecule is called a humoral response, the production of an antibody against a specific foreign molecule (called an antigen). Typically, the antibody's capability of binding the antigen is based on highly complementary structures. That is, the shape of the antibody must contain structures that are the compliment of the structures on the antigen. When the respective structures are fully complimentary, then the two molecules bind tightly.
Antigens are molecules that interact with specific lymphocyte receptors—surface T cell antigen receptors and B cell immunoglobulin receptors. A particular B or T cell binds to a very specific region of the antigen, called an antigenic determinant or epitope. Thus antigens are molecules that bear one or more epitopes which may be recognized by specific receptors in an immune system, a property called antigenicity.
Immunogenicity is the property of stimulating the immune system to generate a specific response. Thus, all immunogens are antigens, but not vice-versa. Although an immune system may recognize an antigen, it does not respond to the antigen unless the antigen is also immunogenic.
An immune response to a particular antigen is greatly influenced by the structure and activity of the antigen itself, as well as myriad other factors. In some cases, the immune system is not able to generate an immune response to a particular antigen, a condition that is called tolerance.
In influencing whether an antigen is immunogenic or immunotolerant, an important characteristic of the antigen is the degree of difference between the antigen and similar molecules within the host. The most immunogenic antigens are those that have no homologs in the host, i.e., those that are most “foreign.” Other factors that promote immunogenicity include higher molecular weight, greater molecular complexity, the proper antigen dose range, the route of administration, the age of the host, and the genetic composition of the host.
As noted above, antigens may have one or more epitopes or binding sites that are recognized by specific receptors of the immune system. Epitopes may be formed by the primary structure of a molecule (called a sequential epitope), or may be formed by portions of the molecule separate from the primary structure that juxtapose in the secondary or tertiary structure of the molecule (called a conformational epitope). Some epitopes are hidden in the three dimensional structure of the native antigen, and become immunogenic only after a conformational change in the antigen provides access to the epitope by the specific receptors of the immune system. This is an important feature and function in the ability of a therapeutic reagent to initiate recognition and response to an antigen, the inducing both a cellular and humoral response to the antigen, and to increasing the antigenicity of a molecule without affecting its immunogenicity.
One of the responses generated by the immune system, a humoral response, involves the production of antibodies. Antibodies beat three major categories of antigen-specific determinants—isotypic, allotypic, and idiotypic—each of which is defined by its location on the antibody molecule. For the purpose of the present invention, we shall only focus on the idiotypic category.
Idiotypic determinants, or idiotopes, are markers for the V region of an antibody, a relatively large region that may include several idiotopes each capable of interacting with a different antibody. The set of idiotopes expressed on a single antibody V region constitutes the antibody idiotype. An antibody (Ab1) whose antigen combining site (paratope) interacts with an antigenic determinant on another antibody V region (idiotope) is called an anti-idiotypic antibody (Ab2). Thus, an antibody includes an antigen binding site, and may include one or more antibody binding sites. There are two types of anti-idiotypic antibodies, sometimes called Ab2α and Ab2β. In one type of anti-idiotype antibody (Ab2β), the combining site perfectly mimics the structure of the antigen epitope recognized by the Ab1 antibody. This type of anti-idiotype is said to represent the internal image of the antigen. By definition, the antigen and this type of anti-idiotype antibody compete for the same binding site on Ab1, and the antigen inhibits the interaction between Ab1 and the anti-idiotypic antibody. The phenomenon of producing an anti-idiotypic antibody having the internal image of the antigen may permit the use of antibodies to replace the antigen as an immunogen.
The second type of anti-idiotype, Ab2α, binds to an idiotope of Ab1 that is distinct from the antigen binding site, and therefore may be characterized in terms of the antigen's inability to prevent the binding of the anti-idiotype to Ab1. For this type of anti-idiotype, Ab1 can bind to both the antigen and the anti-idiotypic antibody. For a graphic representation of these types of antibodies and their interaction, see FIG. 1.
These various interactions based on idiotypic determinants is called the idiotypic network is based on the immunogenicity of the variable regions of immunoglobulin molecules (Ab1) which stimulate the immune system to generate anti-idiotypic antibodies (Ab2), some of which mimic antigenic epitopes (“internal image”) of the original antigen. The presence of internal image antibodies (Ab2) in the circulation can in turn induce the production of anti-anti-idiotypic antibodies (Ab3), some of which include structures that react with the original antigen.
In addition to a humoral response, the immune system may also generate a cellular response mediated by activated T-cells. There are a number of intercellular signals important to T cell activation. Under normal circumstances an antigen degrades or is cleaved to form antigen fragments or peptides. Presentation of antigen fragments to T-cells is the principal function of MHC molecules, and the cells that carry out this function are called antigen-presenting cells (APC: including but not limited to dendritic cells, macrophages, and B cells).
In addition to generating a humoral response, Ab1 and Ab2 have been shown to induce a cellular immune response characterized by proliferative lymphocytes (helper and suppressor lymphocytes), as well as cytotoxic lymphocytes. Therefore, according to the idiotypic network theory, the injection of anti-PSA antibody should result in the induction of a specific cellular and humoral immune response against the PSA molecule. The concept that anti-idiotypic antibodies function as immunogens has been shown by successful immunization against tumoral, bacterial, viral and parasitic antigens in animal models. Generating Ab2 is an indicator of the existence of a robust immune response that inherently reflects the induction of immune system pathways.
The capture and processing of an antigen by APCs is essential for the induction of a specific immune response. The three major APCs are dendritic cells, macrophages and B-lymphocytes; dendritic cells are the most efficient. The injected antibody forms a complex with a circulating PSA, and can be targeted to dendritic cells and macrophages through the Fc-receptors present on these cells. However the high number of Fc receptors on neutrophils may considerably limit this process.
The capture and processing of PSA by B-cells may also occur through the interaction of the membrane bound Ab2 with the anti-PSA/PSA complexes and in a similar manner through the interaction of membrane bound Ab3 with PSA (complexed or not with the anti-PSA antibody).
The present invention is the use of a monoclonal antibody that specifically binds to a circulating prostate-specific antigen at a binding site that results in the production of anti-anti-idiotypic antibodies. A composition according to the present invention induces both a humoral and cellular immune response to PSA. Furthermore, a composition according to the present invention induces or amplifies antigenicity of a tumor-associated antigen without affecting its immunogenicity.
A method according to the invention preferably comprises introducing a sufficient amount of antibody into a host to stimulate a humoral and cellular immune response. The antibody composition may be formulated with an adjuvant or in a liposomal or micellular formulation.
The antibody according to the present invention is useful in generating a humoral and cellular immune response against PSA in a broad segment of the human population with differing HLA class I molecule types. This is in part due to the antibody's specificity for a particular sequence on the PSA molecule.
This sequence is of particular importance for PSA targeted immunotherapy since it contains consensus amino acid motifs for binding to HLA-A2. Of particular interest is the demonstration that the peptides 141-150, 146-154, and 154-163 can generate in vitro human cytotoxic T-lymphocytes capable of lysing human prostate carcinoma cells. Another important observation is the identification of a cleaved bound at position 145-146 of the clipped form of PSA demonstrating that this sequence SEQ ID NO: 1 is particularly sensitive to proteolytic degradation. The selected anti-PSA antibody may therefore upon binding to PSA protect this particular sequence against proteolytic degradation in the endosomal compartment of APCs and enhance the production of immunogenic peptides capable to induce an anti-PSA immune response.
The accompanying drawings show illustrative embodiments of the invention from which these and other of the objectives, novel features and advantages will be readily apparent.
The present invention is a composition comprising a binding agent, such as an antibody, that specifically binds to a prostate specific antigen (PSA), preferably circulating PSA, and even more preferably to an epitope of the PSA molecule corresponding to amino acid sequences 139 to 163. In a preferred embodiment of the invention, the binding agent is a monoclonal antibody.
The present invention is a composition and method of treatment in which the composition includes a binding agent, such as an antibody, that can both induce the production of an anti-anti-idiotype antibody, and can bind with circulating PSA to form a target for antigen presenting cells.
The present invention also comprises a method for treating prostate cancer by administering a composition according to the invention.
The present invention also comprises a composition and method for inducing the production of anti-anti-idiotypic antibodies that specifically bind to a prostate specific antigen.
The present invention also includes a kit containing a composition according to the present invention.
In a preferred embodiment of the invention, a composition comprising a pre-determined antibody that specifically binds to a pre-determined tumor associated antigen is used to bind a soluble antigen produced by the tumor. Once the soluble antigen is bound, the immune system recognizes the antigen as “foreign,” and mounts an immune response against the antigen or against the binding agent bound to the antigen. Antigens that can be made immunogenic are potentially useful to induce or activate an immune response, leading to therapeutic and possibly prophylactic benefits.
As used herein the term “prostate specific antigen” includes the in vivo, ex vivo, or in vitro native protein, the native protein whether purified from a native source or made by recombinant technology, as well as any polypeptide, mutein or portion derived therefrom that is capable of generating an immune response to a native conformationally correct PSA. For example, one can make conservative amino acid substitutions in the molecule without adversely affecting the ability to generate an antibody that will also recognize native PSA.
The compositions and methods of the present invention involve prostate cancer that produces a soluble multi-epitopic tumor-associated antigen (TAA). As used herein soluble is used to describe any antigen that is detectable in a body fluid, i.e., blood, serum, ascites, saliva, or the like. Prostate cancer shed soluble tumor antigens, e.g., tumor antigens shed into the bloodstream, as opposed to a surface antigen or an intracellular antigen; exhibits a multi-epitopic tumor associated antigen, preferably of protein or glycoprotein (e.g., mucin) nature; and can be found at a concentration in the patient's body fluid more than is normally present in healthy controls and such a high level signifies a poor prognosis for the patient, yet has not initiated an immune response. As is well known by one skilled in the art, one method of determining whether the concentration of the TAA is greater than is predictive of recurrence of the disease is by comparing the patient's concentration to that of a healthy control. If the concentration of the TAA is higher than the healthy control, then the patient's concentration is predictive of poor prognosis of the disease.
A binding agent (BA), as used herein, refers to one member of an immunologic pair, e.g., a binding moiety that is capable of binding to a single epitope expressed on the tumor antigen. Exemplary binding agents include, but are not limited to: monoclonal antibodies (“MAb”); chimeric monoclonal antibodies (“C-MAb”); genetically engineered monoclonal antibodies (“G-MAb”); fragments of monoclonal antibodies (including but not limited to “F(Ab)2”, “F(Ab)” and “Dab”); single chains representing the reactive portion of monoclonal antibodies (“SC-MAb”); tumor-binding peptides; humanized antibodies; any of the above joined to a molecule that mediates an effector function; and mimics of any of the above.
The antibody may be a polyclonal antibody or a monoclonal antibody. When the subject is a human subject, the antibody may be obtained by immunizing any animal capable of mounting a usable immune response to the antigen, such as a mouse, rat, goat sheep, rabbit or other suitable experimental animal. In the case of a monoclonal antibody, antibody producing cells of the immunized animal may be fused with “immortal” or “immortalized” human or animal cells to obtain a hybridoma which produces the antibody. If desired, the genes encoding one or more of the immunoglobulin chains may be cloned so that the antibody may be produced in different host cells, and if desired, the genes may be mutated so as to alter the sequence and hence the immunological characteristics of the antibody produced.
Fragments, or fragments of binding agents, may be obtained by conventional techniques, such as by proteolytic digestion of the binding agent using pepsin, papain, or the like; or by recombinant DNA techniques in which DNA encoding the desired fragment is cloned and expressed in a variety of hosts. Irradiating any of the foregoing entities, e.g., by ultraviolet light will enhance the immune response to a multi-epitopic antigen under similar conditions. In a preferred embodiment of the invention, effector functions that mediate CDC or ADCC are not required.
The present invention includes substitutions and deletions within the amino acid structure of the antibody provided that the modifications result in a functionally equivalent antibody with enhanced immunogenicity.
In an embodiment of the invention, a composition includes an antibody that specifically binds to circulating prostate-specific antigen. In an embodiment of the invention, the antibody binds to an epitope of PSA corresponding to amino acid sequences 139-163 of the PSA molecule. More specifically, the antibody binds to an epitope corresponding to the following amino acids: EEFLTPKKLQCVDLHVISNDVCAQV (Seq. SEQ ID NO. 1). In a most preferred embodiment, a binding agent according to the invention binds to an epitope of PSA corresponding to amino acid sequences 135 to 150 of the PSA molecule. In accordance with the present invention, other binding agents may be useful in the practice of this invention. For example, other suitable binding agents, such as monoclonal antibodies, include those that bind near the N-terminus of the PSA molecule.
The antibody of the present invention may be formulated into a pharmaceutical composition in combination with a pharmaceutically acceptable carrier for use as an immunogen in a mammal, preferably a human or primate. The composition may further comprise one or more other constituents to enhance the immune response which include but are not limited to biological response modifiers such as dendritic cell growth factors, such as Flt 3; interleukin 2, interleukin 6, interleukin 12, interferon, tumor necrosis factor, GM-CSF and cyclophosphamide. The composition may also comprise at least one HLA class I molecule, or a cell expressing at least one HLA class I molecule in combination with one or more antibodies or fragments thereof. Adjuvants include, for example, RIBI Detox, QS21, alum and incomplete Freund's adjuvant. Liposomal formulations can also be used.
The composition may include one or more adjuvants, one or more carriers, one or more excipients, one or more stabilizers, one or more imaging reagents, and/or physiologically acceptable saline. Generally, adjuvants are substances mixed with an immunogen in order to elicit a more marked immune response. Control vaccinations without the adjuvant resulted in humoral immune responses. The composition may also include pharmaceutically acceptable carriers. Pharmaceutically accepted carriers include but are not limited to saline, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic substances suitable for delivery to a patient may be included in the compositions of the present invention. The compositions may be solutions suitable for administration, and are typically sterile and free of undesirable particulate matter. The compositions may be sterilized by conventional sterilization techniques.
A composition according to the invention may be administered to a mammal in an amount effective in generating a PSA specific humoral and cellular immune response. The efficacy of the antibody as an immunogen may be determined by in vivo or in vitro parameters as are known in the art. These parameters include but are not limited to antigen specific cytotoxicity assays, regression of PSA+ tumors, inhibition of PSA+ cancer cells, production of cytokines and the like.
As used herein, “administering” refers to any action that results in exposing or contacting a binding agent, such as an antibody, with a pre-determined antigen, cell, cells, or tissue, typically mammalian. As used herein, administering may be conducted in vivo, in vitro, or ex vivo. For example, a composition may be administered by injection or through an endoscope. Administering also includes the direct application to cells of a composition according to the present invention.
In accordance with a method of the invention, the binding agent must be capable of binding a pre-determined binding site or receptor, and may be administered to the patient by any immunologically suitable route. For example, the binding agent may be introduced into the patient by an intravenous, subcutaneous, intraperitoneal, intrathecal, intravesical, intradermal, intramuscular, or intralymphatic routes. The composition may be in solution, tablet, aerosol, or multi-phase formulation forms. Liposomes, long-circulating liposomes, immunoliposomes, biodegradable microspheres, micelles, or the like may also be used as a carrier, vehicle, or delivery system. Furthermore, using ex vivo procedures well known in the art, blood or serum from the patient may be removed from the patient; optionally, it may be desirable to purify the antigen in the patient's blood; the blood or serum may then be mixed with a composition that includes a binding agent according to the invention; and the treated blood or serum is returned to the patient. The clinician may compare the anti-idiotypic and anti-isotypic responses associated with these different routes in determining the most effective route of administration. The invention should not be limited to any particular method of introducing the binding agent into the patient.
In accordance with the methods of the present invention, a composition comprising the binding agent may be administered in an amount sufficient to recognize and bind the prostate tumor associated antigen. In a preferred embodiment of the invention, the dosage is sufficient to generate or elicit an immune response against the TAA. An immunologically or therapeutically effective or acceptable amount of binding agent is an amount sufficient to bind a predetermined antigen in vivo or ex vivo, and is capable of eliciting an immune response to the antigen. The response inhibits or kills tumor cells that carry and present a newly accessible epitope, thereby ameliorating or eliminating the disease or condition that produces the antigen. The immune response may take the form of a humoral response, a cell-mediated response, or both. In a preferred embodiment of the invention, the dosage of the monoclonal antibody is less than the dosage required to elicit ADCC or CDC.
The concentration or dosage of the protein in the composition can vary widely, e.g., from less than about 0.01% to about 15 to 20% by weight. As noted above, the composition is administered in an amount sufficient to stimulate an immune response against the antigen. Amounts effective for this use will depend in part on the severity of the disease and the status of the patient's immune system. Generally, the composition will include about 0.1 μg to about 2 mg or more of protein agent per kilogram of body weight, more commonly dosages of about 1 μg to about 200 μg per kilogram of body weight. The concentration will usually be at least 0.5%; any amount may be selected primarily based on fluid volume, viscosity, antigenicity, etc., in accordance with the particular mode of administration.
Administration may be more than once, preferably three times over a prolonged period. As the compositions of this invention may be used for patient's in a serious disease state, i.e., life-threatening or potentially life-threatening, excesses of the binding agent may be administered if desirable. Actual methods and protocols for administering pharmaceutical compositions, including dilution techniques for injections of the present compositions, are well known or will be apparent to one skilled in the art. Some of these methods and protocols are described in Remington's Pharmaceutical Science, Mack Publishing Co. (1982). Administration may also include ex vivo administration protocols, e.g., removing a portion of a patient's body fluid, contacting in vitro the body fluid with the therapeutic composition, and then returning the treated body fluid to the patient.
A binding agent may be administered in combination with other binding agents, or may be administered in combination with other treatment protocols or agents, e.g., chemotherapeutic agents.
The therapeutic efficacy of the administered anti-PSA antibody is believed to depend on two different mechanisms of action, although the invention is not to be restricted to the recited mechanisms. The first mechanism is the induction of a specific immunity (humoral immunity) to PSA via the idiotypic network. The second mechanism involves cellular immunity—the administered anti-PSA antibody binds to circulating PSA to form a complex suitable for binding antigen presenting cells (APCs). Of particular interest, this mechanism of action generates a multi-epitopic immune response against the PSA. The targeting of Ar 47.47 at the tumor site upon i.v. injection may also favor the migration and accumulation of immunocompetent cells at the tumor site. The secretion of cytokines by these immune cells may in turn stimulate the production of a protective immune response against various tumor antigens.
In accordance with another embodiment of the invention, the structure of the antibody may be modified in order to enhance its capture by dendritic cells. Exemplary modifications include but are not limited to mannosylation of the anti-PSA antibody or conjugation to an antibody specifically directed against a murine dendritic cell surface marker. Unlike the Fc receptors, the mannose receptor is recycled to the cell surface after endocytosis and can therefore allows internalization of ligands in successive rounds. Indeed, mannosylated proteins are presented with 100-fold higher efficiency than un-glycosylated proteins by dendritic cells.
Production of AR47.47
Hybridoma clones secreting anti-PSA antibodies were produced by fusion of the murine myeloma cells Sp2/O with the splenocytes of a Balb/c mouse immunized with human PSA (FIG. 3).
Immunogen: PSA purified from human seminal plasma was obtained from Scripps Laboratories, San Diego, Calif. [Cat # P0723 (purity, about 50%); Cat # P0725 (purity, 99%)].
Mice: Male Balb/cCr/AltBM mice, 6 weeks of age, were purchased from Jackson Laboratories and housed on 7th floor Dentistry/Pharmacy Building (HSLAS facilities).
Immunization Protocol: Mice were immunized i.p. with 30 μg of PSA (Cat # P0723) in complete Freund adjuvant. The mice received subsequently two i.p. injections at 10 days interval with 30 μg of PSA (Cat # P0723). The first booster was performed in incomplete Freund adjuvant whereas the 2nd booster was performed in PBS. The serum levels of anti-PSA antibodies were measured by ELISA on PSA coated plates (ELISA # 1) and the mouse with the higher serum level was subjected to a final intra-splenic boost with 30 μg of PSA (Cat # P0725) in PBS.
Fusion: 3 days after the final intra-splenic boost the hybridomas were generated by fusion of spleen cells with the murine myeloma cell line Sp2/0-Ag14 (ATCC CRL-1581).
First screening: Hybridoma clones secreting anti-PSA antibodies were first detected by ELISA on PSA coated plates (ELISA #1). We selected 80 positive clones from this first screening. Those clones are currently kept frozen in liquid nitrogen tank.
Second screening: The positive clones were further screened against PSA complexed to alpha anti-chymotrypsin (PSA-ACT) (ELISA #2); PSA non complexing to alpha anti-chymotrypsin (ELISA #4); Plasmatique kallikrein (ELISA #5); and PSA peptide 139-163 (ELISA #6)
The reactivity of the AR47 clones towards the different forms of PSA and towards kallikrein is reported in Example 2. The isotype of the antibodies secreted by AR47 clones is reported in Example 3.
The criteria of selection of a clone for additional studies was based on the reactivity towards PSA and PSA-ACT (the two main forms of immunoreactive PSA in human serum), the reactivity towards PSA peptide 139-163 (PSA sequence involved in cellular immunity), and the isotype of the secreted antibodies. Based on these criteria, the hybridoma clone AR47.47 was selected.
Adaptation from HAT Medium to Standard Medium:
AR47 clones arising from 96 well plates and grown in HAT medium were first transferred into 24 well plates containing 2 ml of HT medium/well and then, once confluence was reached, transferred into 6 well plates containing 6 ml of HT medium/well. The adaptation of the cells from HT medium to standard medium was performed gradually.
HAT medium: RPMI 1640, 10% FBS, HAT supplement, OPI supplement, 0.3 mg/ml glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin
HT medium: RPMI 1640, 10% FBS, HT supplement, OPI supplement, 0.3 mg/ml glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin Standard medium: RPMI 1640, 10% FBS, 0.3 mg/ml glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin.
Ascites can be produced in mice pre-treated with pristane (0.5 ml/mouse) and injected with AR47.47R6R6. The concentration of AR47.47 in ascites was calculated to be in the range of 4 to 6 mg/ml.
Prostate specific antigen (PSA) represents an attractive target for the immunotherapy of prostate cancer. This glycoprotein is almost exclusively synthesized by the prostatic gland and is currently used for the diagnosis and monitoring of prostate cancer patients. However, since PSA is recognized as a self-antigen, it is essential for effective immunotherapy to develop innovative strategies capable of triggering the immune system and induce a protective immunity against PSA expressing cells. This example demonstrates the use of an antibody to elicit an anti-idiotype cascade associated with an antigen specific anti-tumor immune response. A large panel of anti-PSA monoclonal antibodies have been produced in our laboratory and these antibodies were evaluated for their potential therapeutic efficacy against prostate cancer. We have demonstrated that the immunization of mice with a selected anti-PSA antibody can induce a specific immunity against PSA itself These results therefore emphasize the potential use of anti-PSA antibodies for the immunotherapy of prostate cancer.
Hybridoma clones secreting anti-PSA antibodies were produced by fusion of the murine myeloma cells Sp2/O with the splenocytes of a Balb/c mouse immunized with human PSA. An exemplary clone, AR47.47, binds to an epitope of PSA corresponding to amino acid sequences I39-I63 of the PSA molecule. It has now been shown that AR 47.47 also recognizes amino acid sequences I35-I50, produces a stronger signal, and may be the minimum sequences required for binding.
The hybridoma clone AR47.47 was deposited on Apr. 29, 1998 with the American Type Culture Collection (ATCC), located at 10801 University Blvd., Manassas, Va. 20110-2209, in accordance with the Budapest Treaty. The AR47.47 hybridoma clone was assigned the ATCC designation number HB-12526.
The first criteria of selection used to identify the anti-PSA antibody was the ability of this antibody to interact with circulating PSA. Circulating PSA is found either in a free form or complexed to anti-proteases such as α-anti-chymotrypsin and α2-macroglobulin. To screen for clones we used three different forms of PSA: free PSA; PSA complexed to α-anti-chymotrypsin (PSA-ACT); and free PSA non complexing to α-anti-chymotrypsin (PSA-nc). Free PSA corresponds to PSA directly purified from human seminal fluid. Co-incubating free PSA with purified ACT results in the formation of PSA-ACT and PSA-nc. PSA-nc can be separated by gel filtration chromatography. It is believed that PSA-nc may represent the free form of PSA present in the circulation. Complexing of PSA with α2-macroglobulin results in the total encapsulation of PSA. As a consequence, this form of PSA is no longer detectable by monoclonal anti-PSA antibodies. We therefore did not use this form of circulating PSA for the screening.
PSA belongs to the kallikrein family and a high degree of structural homology is found between PSA and the kallikreins HKI and HK2. The absence of cross reactivity of the anti-PSA antibody with kallikrein isolated from human plasma was used as second criteria for selection.
The hybridoma clone AR47.47 responded to the criteria described above, a strong immunoreactivity was observed with the three forms of PSA used for the screening whereas no cross reactivity was observed with human plasmatic kallikrein. The hybridoma clone AR47.47 was cloned twice by limiting dilution and the second generation clone AR47.47R6R6 was chosen for further studies. Clone AR47.47R6R6 was adapted to standard medium (RPMI 10% FBS) and a cell bank was formed. The absence of mycoplasma contamination was verified by using the Boehringer Manheim mycoplasma test. Clone AR47.47R6R6 has been deposited in the American Type Culture Collection, and has received Accession No. H-B12526.
Immunization in DBA mice with a binding composition according to the invention (AR47.47) was examined for the induction of a specific PSA immunity via the idiotypic network (i.e. induction of Ab3 antibodies). Anti-PSA antibodies (Ab3) could be detected in the serum of animals immunized with AR 47.47, a minimum of two injections of AR 47.47 was required for Ab3 production. No reactivity towards PSA was detected for the control groups (mice immunized with an isotype matched control antibody not related to PSA and mice receiving PBS injections).
AR 47.47 is directed towards a PSA epitope comprised between the sequence I39-I63 of the PSA molecule. The anti-PSA antibodies produced by AR 47.47 immunized mice can specifically interact with the PSA peptide I39-I63, showing that at least part of the Ab3 produced are identical in term of specificity to AR 47.47. These results demonstrate that the immunization with AR 47.47 can induce a specific anti-PSA immunity in the host.
Clone Culture and Productivity
Growth Characteristics in Standard Medium
Using standard medium (RPMI 1640, 10% FBS, 0.3 mg/ml glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin), AR 47.47R6R6 hybridoma cells were seeded at 5×104 cells/ml in 6 well plates and cultured for several days. Each day the cell concentration was defined by counting the cells manually with an hematocytometer, the cell viability was determined by trypan blue exclusion, and the antibody concentration in the culture media was measured by ELISA (ELISA #8). Three independent experiments were performed.
The growth characteristics of the AR47.47R6R6 hybridoma clone are shown in FIG. 4 and the following table:
The AR47.47R6R6 clone has a generation time of 22 hours and can grow to a maximum of 0.6 millions cells/ml. Highest antibody level in the culture media was calculated to be approximately 60 μg/ml.
AR47.47 hybridoma cells were adapted to serum free medium (SFM, Gibco) and protein free medium (UltraDOMA-PF, Biowhitaker). AR47.47R6R6 hybridoma cells were adapted to serum free medium (SFM, Gibco, cat #12045-084) and protein free media (UltraDOMA-PF, Bio Whittaker, cat #15-7-27 and 08-242F). AR47.47R6R6 was easily adapted to the protein free medium UltraDOMA. In this medium, the cells were best maintained when seeded at a concentration lower than 0.1 millions cells/ml.
The growth characteristics of AR47.47R6R6 in standard media (RPMI 10% FBS), in RPMI 5% FBS, and in UltraDOMA are compared in the following table:
The adaptation of AR47.47R6R6 to serum free medium (SFM, Gibco) was in contrast considerably more difficult. The cells did not survived if diluted at a concentration lower than 1×105 cells/ml. The cells were best maintained at a cell concentration comprised between 2-4×105 cells/ml. The growth characteristics in this medium were not studied.
AR 47.47 was purified by affinity chromatography from either ascites or from cell culture medium. The affinity column used is the Gamma Bind Plus Sepharose from Pharmacia Biotech. The purification of the antibody was performed according to the manufacturer instruction protocol. The chromatography was monitored by measuring the absorbency at 280 nm (total protein concentration) and by testing the different fractions for the presence of anti-PSA antibody (ELISA #1). The fractions containing purified antibody were concentrated and dialyzed against PBS. The purity of the antibody was analyzed by SDS-PAGE. The concentration of the antibody in the starting materials and in the purified fractions was determined by ELISA (ELISA #8) and by measuring the absorbency at 280 nm (the conversion of the absorbency value to a concentration value was made using an ε=1.46).
AR47.47 is an IgG1 antibody and interacts specifically with free PSA as well as with PSA complexed with ACT. Synthetic linear PSA peptides were used to identify the epitope recognized by AR47.47 (FIG. 5). Direct binding as well as competitive experiments demonstrate that the epitope recognized by AR47.47R6R6 is comprised between the PSA sequence 139-163. AR47.47 does not recognize human plasmatic kallikrein. The isotype of AR47.47R6R6 was determined in an ELISA kit (Southern Biotechnology), and is a murine IgG1,k antibody. AR47 antibodies specifically recognize PSA, PSA-ACT, and PSAnc.
The domain of the PSA molecule comprised between the sequence 140-163 is of particular interest for the development of an immunotherapeutic product for prostate cancer. Indeed, the following PSA peptides; 141-150, 146-154, 154-163, bind to HLA-A2 molecules and cytotoxic T lymphocytes specific for these peptides have been generated in vitro.
Using a direct binding assay with the N-biotinylated PSA peptide 139-163, we have demonstrated that AR47.47 is specifically directed against this sequence (ELISA #5). Only AR47.47 binds to the PSA peptide 139-163. The specificity of AR 47.47 for the PSA sequence 139-163 was further confirmed by a competitive binding assay (ELISA #7). The binding of AR47.47 to immobilized PSA can be inhibited in a dose dependent manner by N-biotinylated PSA peptide 139-163(SEQ ID. NO: 1). In an attempt to identify more precisely the minimal sequence recognized by AR 47.47, we have studied, using the same competitive assay (ELISA #7), the inhibition of the binding of AR47.47 to PSA by the PSA peptides 141-150(SEQ ID. NO: 2), 146-154(SEQ ID. NO: 3), 154-163(SEQ ID. NO. 4):
The 9 amino-acid short peptide sequences were not able to inhibit the binding of AR 47.47 to PSA. The absence of inhibition can be explained by AR 47.47 recognizing a conformational epitope, and/or AR 47.47 recognizing a linear epitope but the peptide sequences used are not representative of the minimal binding sequence of AR 47.47 for PSA (for example AR 47.47 may recognizes the region 139-148 of the PSA molecule). It is worth mentioning that all the peptide sequences used possess a cysteine. It is likely that the peptides will dimerize immediately after solubilization by formation of a disulfide bridge between two cysteine residues. The dimerized peptides may not be recognized by AR47.47. It has been shown, however, that various antibodies recognize an epitope near the N-terminus of the PSA molecule, and these antibodies induce a therapeutically beneficial immune response.
The measurement of the affinity of AR 47.47 for PSA was determined using either radiolabeled AR 47.47 (RIA #1) either radiolabeled PSA (RIA #2). The Kd values calculated from independent experiments are reported in the following table:
The PSA sequence recognized by AR 47.47 does not contain tyrosine and therefore is likely not to be affected by the iodination process. The slightly higher affinity of AR 47.47 calculated when radiolabeled PSA is used in comparison of radiolabeled AR 47.47 may suggest that the affinity of the antibody is slightly diminished by the iodination process.
The affinity of AR 47.47 for PSA-ACT was performed using radiolabeled AR 47.47 (RIA #3). Two independent experiments were performed. The affinity constants calculated from the Scatchard plots were 1.6 nM and 1 nM respectively. The affinity of AR47.47 for both PSA and PSA-ACT therefore appears to be close.
The affinity of AR 47.47 for PSA peptide 139-166 was performed using radiolabeled AR 47.47 (RIA #4). Three independent experiments were performed. The affinity constants calculated from the Scatchard plots demonstrate similar affinity of AR 47.47 for both PSA and PSA peptide 139-163.
The fragmentation of AR47.47R6R6 antibody into F(ab)2 fragments was realized by enzymatic digestion using immobilized ficin (Pierce) as described by the manufacturer instructions. The separation of the Fab fragments from the Fc fragments was performed by affinity chromatography on Protein A column. The non-bound fractions washed out of the column contain the fab fragments whereas the bound fractions contained the Fc fragment and eventually the remaining non-digested antibody.
The fractions eluted from the Protein A column were first tested for their immunoreactivities with anti-Fc and anti-Fab antibodies (ELISA #9 and 10). The results obtained indicate that the first fractions directly eluted from the column contain as expected Fab [either Fab or F(ab)2] fragments and were not contaminated with Fc fragments. We further demonstrated that these Fab fragments were still able to bind PSA (ELISA #11, RIA #5): SDS PAGE analysis of AR 47.47 purified from GammaBind+column.
Two murine PSA-transfected cell lines (Line-1-PSA; P815-PSA) were obtained from Dr. E. M. Lord (University of Rochester, NY). Line-1 is a Balb/cByJ (H-2d) mouse lung carcinoma cell line and P815 is a DBA/2 (H-2d) mastocytoma cell line. The transfected cell lines Line-1-PSA and P815-P SA are stably transfected and secrete high level of PSA in the culture medium (FIG. 6). The levels of PSA expression by these transfected cell lines is comparable to what has been observed in the dihydrotestosterone-induced human prostate cell line LnCAP [Wei et al; Cancer Immunol Immunother, 42:362-368 (1996)].
The expression of MHC class I and MHC class II molecules was studied by FACS analysis using the commercially available antibodies directed against H-2KdDd (MHC class I) and I-Ad (MHC class II). P815-PSA cells grown in vitro or isolated from solid tumor (from DBA mouse) express high level of MHC class I molecules but only low levels of MHC class II molecules. When grown in vitro, Line 1-PSA expressed very low levels of MHC class I and II molecules. However, the expression of MHC class I and II molecules could be induced by treatment of the cells for 5 days with 3% DMSO.
Two syngeneic tumor models were established. The first animal model consists of Balb/cByJ mice injected intravenously with 0.1 million Line-1-PSA tumor cells. After 3-4 weeks the mice were sacrificed and the tumor burden was calculated by measuring the number of tumor foci in the lung and by measuring the weight of the lungs.
The second animal model consists of DBA/2 mice injected subcutaneously with 0.5 million P815-PSA cells. After 2 weeks the tumors were palpable and measured at regular intervals with a caliper. Four weeks after the inoculation of the tumor cells, the mice were sacrificed, the tumors are dissected and weighed.
Anti-idiotypic Induction of PSA Immunity in Mice
Mice were used to determine whether immunization with anti-PSA antibodies can induce a specific immunity against PSA via activation of the idiotypic network. The goal of this experiment was to demonstrate that the immunization of mice with anti-PSA antibodies (Ab1) can stimulate the immune system to generate anti-idiotypic antibodies (Ab2=surrogate antigen), and anti-anti-idiotypic antibodies (Ab3) capable of reacting with the original antigen.
These experiments used a commercially available antibody as a model anti-PSA antibody (RLSD09; ATCC HB-8525). The purified antibody was conjugated to Keyhole Limpet Hemocyanin (KLH) to enhance its immunogenicity. The anti-PSA antibodies conjugated to KLH were still capable of binding to PSA, indicating that the idiotype of the antibodies were not masked by the conjugation procedure. B43.13 antibody, a mouse monoclonal antibody of the same isotype as the PSA antibody (IgGI) was used as the control. B43.13 antibody is specifically directed against the CA125 ovarian tumor antigen and does not cross react with PSA. In addition FACS analysis verified that the B43.I3 antibody does not bind at the cell surface of Line-1-PSA or P81 5-PSA.
Mice were subdivided into three groups of five mice each. The first group of mice was immunized with anti-PSA antibody conjugated to KLH. The second group of mice was immunized with the control B43.13 antibody conjugated to KLH. The third group of mice received PBS injection. Injections were performed i.p. at 10 days intervals with complete Freund adjuvant for the first injection and incomplete Freund adjuvant for the second injection.
Ab2 is a surrogate antigen capable of mimicking the PSA epitope recognized by the injected anti-PSA antibody. A competitive inhibition assay was established to measure the serum level of Ab2. This assay was performed 5 days after the second injection. An inhibition was observed after incubation in the presence of mouse sera from mice immunized with anti-PSA antibody, but not when sera from mice immunized with control antibody or PBS were used. These results indicate that the immunization of Balb/c mice (FIG. 7A)and DBA mice (
Ab3 corresponds to the anti-PSA antibodies produced by the host. The Ab3 level in the mouse serum was measured by ELISA on PSA coated plate, performed 5 days after the second injection (FIGS. 8A-8B). The anti-PSA antibodies measured by this assay can not correspond to the injected antibodies. Anti-PSA antibodies could be detected in the mouse serum of DBA mice and Balb/c mice immunized with anti-PSA antibody. These results demonstrate that an anti-PSA immune response can be induced in mice by immunization with an anti-PSA monoclonal antibody.
Effect of Anti-PSA Immunization on Tumor Development
Balb/c mice were used to determine whether immunization with anti-PSA antibodies can protect the animals against a subsequent tumor challenge. Balb/c mice were divided into 3 groups of 5 mice each. The first group was immunized with anti-PSA antibody RLSD09 conjugated to KLH, the second group was immunized with control antibody B43 conjugated with KLH, the third group received PBS injections. A total of 4 injections were given for each group using 50 μg of antibodies for each injection. The tumor cells Line-1-PSA were injected intravenously between the third and fourth injections. Nineteen days after tumor inoculation, the mice were sacrificed, the number of tumor foci in the lungs and Ab3 levels in the serum were determined.
The tumor burden in the group of mice immunized with anti-PSA MAb was considerably lower compared to the group of mice immunized with control antibody. Of particular interest is the demonstration, in the group of mice immunized with anti-PSA MAb, of a negative correlation between Ab3 levels and the number of tumor foci in the lungs (FIG. 9).
Effect of Immunization on the Induction of a Specific PSA Immunity
We have studied in DBA mice whether the immunization with a monoclonal antibody that specifically binds to PSA (AR47.47) can result in the induction of a specific PSA immunity via the idiotypic network (i.e. induction of Ab3 antibodies). Anti-PSA antibodies (Ab3) could be detected in the serum of animals immunized with AR 47.47, a minimum of two injections of AR 47.47 was required for Ab3 production. No reactivity towards PSA was detected for the control groups (mice immunized with an isotype matched control antibody not related to PSA and mice receiving PBS injections). See
Ar 47.47 is directed toward a PSA epitope comprised between the sequence 139-163 of the PSA molecule. The anti-PSA antibodies produced by AR 47.47 immunized mice can specifically interact with the PSA peptide 139-163 (
The syngeneic tumor animals models used for these studies are:
Therapeutic antibodies: AR47.47R6R6 a murine monoclonal antibodies of the IgG1 isotype specifically directed against PSA
Control antibodies: murine monoclonal antibodies of the IgG1 isotype: CA 125 MAb directed against the tumor antigen CA125 (AltaRex Corp.) MOPC-21 a mouse myeloma immunoglobulin of unknown specificity (Sigma)
The absence of interaction between control antibodies and PSA was verified by ELISA.
The binding of the anti-PSA antibodies and control antibodies to the PSA-transfected tumor cell lines was studied by FACS analysis. None of the anti-PSA antibodies studied bound to the surface of PSA transfected tumor cells grown in vitro. The control antibodies did not bind to the surface of Line-1-PSA tumor cells. A very low binding of the two control antibodies was observed for P815-PSA cell line.
Anti-PSA antibodies and control antibodies were conjugated to KLH in order to enhance their immunogenicity in mice (glutaraldehyde conjugation, 1 mole IgG/0.2 mole KLH). The PSA binding activity of AR 47.47 after KLH-conjugation was considerably reduced by the conjugation process (90%).
Induction of a Specific Anti-PSA Immune Response in Mice by Immunization with AR 47.47.
The elicitation of a specific PSA immune response upon immunization with AR 47.47 and induction of a specific idiotypic network was studied both in Balb/c mice and DBA mice. In this set of experiments (experiments #5, 6, 7, 9, 11, 12), the mice were first treated with either AR 47.47, control MAb or PBS, and then inoculated with PSA-transfected tumor cells. The results correspond to the measurement of the anti-idiotypic (Ab2) and anti-anti-idiotypic (Ab3) immune response against AR 47.47 before tumor inoculation.
The detection of anti-idiotypic antibodies (Ab2) was performed by ELISA using plates coated with AR 47.47 F(ab) fragments (produced by ficin digestion). The presence of Ab2 in the mouse sera of AR 47.47 immunized mice was demonstrated in experiments 7, 9, 11 and 12.
Two different ELISA assays were performed to detect the presence of Ab3. The first assay detects the binding of Ab3 on PSA coated plate. Using this assay, we have demonstrated in experiments #5, 6 and 7 the presence of Ab3 in the sera of mice immunized with AR 47.47. The second assay employs the PSA peptide known to be recognized by AR 47.47. This assay gave positive signal for the mice immunized with AR 47.47 in experiment 7. This second assay however has not been standardized at this time and the results should be analyzed with caution since in many cases the positive control (performed with AR 47.47) showed negative signal. Since the PSA peptide used for this assay contains cysteine residues we believed that a cyclisation or polymerization of the peptide occurs after solubilization and/or storage of the peptide. Such effect may impairs the binding of the peptide to streptavidin coated plate or to specific antibodies (i.e., AR 47.47 or Ab3).
A competitive ELISA assay employing the PSA peptide recognized by AR 47.47 was also performed. The inhibition of the binding of PSA peptide to AR 47.47 immobilized on ELISA plate is theoretically dependent on the presence of both Ab2 and Ab3 in the mouse sera. A competitive inhibition was observed for experiments #7, 9, 11 and 12.
These results demonstrate that an anti-idiotypic network resulting in the formation of anti-PSA antibodies by the host could be induced in mice (Balb/c and DBA) upon immunization with AR 47.47.
Effect of AR 47.47 on Tumor Development
The ability of AR 47.47 to inhibit the development of PSA-transfected tumors was studied in both Balb/c mice (experiment #5, 7, 11) and DBA mice (experiment 6, 9, 12). In this set of experiments the tumor inoculation occurs after treatment of the mice with either AR 47.47, control MAb or PBS. The immunization of the mice was continued after tumor inoculation.
For each experiment, the following parameters were measured: Ab2; Ab3; Ab2+Ab3; tumor burden. The results obtained for each experiment are shown in the following tables:
Effect of AR47.47-KLH immunization on Line 1-PSA tumor development in Balb/c mice
Effect of AR47.47-KLH immunization on P815-PSA tumor development in DBA mice
A lower tumor burden in the group of mice treated with AR 47.47 was observed in 1 experiment out of 3 for Balb/c mice tumor model and in 2 experiments out of 3 for the DBA mice tumor model. The same ELISA assays described in the previous paragraph were used to measure the humoral immune response in mice after tumor inoculation. The presence of Ab2 was detected in the groups of mice immunized with AR 47.47 in all experiments. The presence of Ab3 was detected in all groups of mice in all experiments. The positive signal obtained for Ab3 in the control groups (PBS and control mab) is not surprising since the release of human PSA by the growing tumor in vivo will induce an anti-PSA immune response.
Effect of AR 47.47 on Tumor Progression
The ability of AR 47.47 to inhibit the progression of PSA-transfected tumors was studied in both Balb/c mice (experiment #8, 13) and DBA mice (experiment 10, 14). In this set of experiment the tumor inoculation occurs before treatment of the mice with either AR 47.47, control MAb or PBS.
For each experiment, the following parameters were measured: Ab2; Ab3; Ab2+Ab3; and tumor burden.
The results obtained for each experiment are resumed in the following tables:
Effect of AR47.47-KLH immunization on Line-1 PSA tumor progression in Balb/c mice
No therapeutic effect on tumor progression was observed for AR47.47. However it should be taken into consideration that the growth rate of tumors in mice far exceed the time required for the development of an appropriate therapeutic immune response capable to eradicate or slow tumor progression. Also the growth rate of tumor in mice do not reflect what is happening in humans since prostate cancer is considered to be a slow progressing disease.
Possible Mechanism of Action
Although the present invention has been described in terms of a particular preferred embodiments, it is not limited to those embodiments. Alternative embodiments, examples, and modifications which would still be encompassed by the invention may be made by those skilled in the art, particularly in light of the foregoing teachings.

domain: medical


Former Indiana Republican Sen. Richard Lugar, who died at 87 on Sunday of a rare neurological disorder, never got to be president, though he made an unsuccessful run for the job in 1996.
He never even got to be vice president. While he was on President Reagan's short list in 1980, Reagan ultimately chose George H.W. Bush as his running mate. While President Bush also considered Lugar for the post, he wound up tapping another politician for the job -- Indiana's junior senator Dan Quayle.
Lugar was not awarded the Nobel Peace Prize either. But he should have won.
For despite his modesty, quiet reserve and self-deprecating manner, Lugar helped do more than most politicians combined to help protect the country, indeed, the world from the scourge of nuclear, chemical and biological weapons. He leaves an extraordinary national security legacy.
As the Soviet Union was collapsing, Lugar crossed party lines to work with Georgia Sen. Sam Nunn, then chairman of the Senate Armed Services Committee, on what seemed a radical, risky idea to prevent Moscow's Weapons of Mass Destruction (WMD) from falling into the wrong hands. In 1991, they won Congressional approval of what became known as the Cooperative Threat Reduction program (CTR), or "Nunn-Lugar," for short.
There was no shortage of critics back then of their counter-intuitive initiative: Washington would pay its main former enemy – Russia and the other former Soviet republics -- to help them secure, dismantle, or destroy its vast arsenal of an estimated 35,000 nuclear weapons, hundreds of tons of plutonium and highly enriched uranium, and chemical weapons arsenals and biological weapons stockpiles at dozens of facilities spread across 11 time zones.
William H. Tobey, a former deputy administrator of the National Nuclear Security Administration from 2006 to 2009, remembers what American weapons experts saw when they first arrived at Russian nuclear sites in the early 1990s. They found fallen fences, decrepit buildings, and broken morale among young, underpaid, ill-trained, bored, and often drunk Russians guarding weapons that could readily destroy the world.
Since 1992, American scientists and weapons technicians, working under CTR, at a cost of over $2 billion, have installed cameras that provide constant remote monitoring of the most sensitive materials, metal and radiation detectors to protect against unauthorized intrusions. They have reinforced doors and windows, installed locks, built bulletproof guard facilities, perimeter fences and robust barriers at vehicle entry points. They have laid hundreds of "man traps" -- heavy turnstiles that require authorized access and proper identification before opening. The work was often hard and conditions difficult. The temperature at one facility I visited in Siberia dropped below -65 degrees Fahrenheit in winter and rose above 100 degrees in summer.
CTR also facilitated the arrangements that led Ukraine, Belarus, and Kazakhstan to forego the nuclear weapons Moscow had based on their territory. As a result, all three countries are now nuclear weapons free. The program has deactivated more than 7,600 strategic nuclear warheads – more than the nuclear arsenals of France, Britain and China combined, according to a 2014 Pentagon study. It has destroyed more than 2,500 nuclear-capable missiles, 1,187 missile launchers, 33 submarines, and 155 bombers. More than 1,675,097 rounds of chemical munitions have been destroyed, along with some 4,129 metric tons of chemical weapons.
When Nunn retired in 1997, Lugar continued promoting CTR and its expansion to countries outside the former Soviet empire. Building on the expertise acquired in dealing with Soviet WMD, CTR has evolved into a promoter of non-proliferation cooperation and a center of expertise that has reduced WMD threats throughout the world. It was instrumental, for instance, in securing and neutralizing chemical weapons stockpiles in Albania and Libya.
In 2005, Richard Lugar took then-junior Illinois Sen. Barack Obama on his first trip to Ukraine and Azerbaijan to visit dismantled weapons sites. President Obama later credited that trip, and Lugar, with having reinforced his determination to reduce the size of America's nuclear arsenal.
In 2013, Obama awarded Lugar the Presidential Medal of Honor, the nation's highest civilian honor. "His legacy," said Obama, "is the thousands of missiles and bombers and submarines and warheads that no longer threaten us because of his extraordinary work."
Later years were not kind to Lugar or his work. In 2012, he lost the Republican nomination when seeking his seventh Senate term. Though state Treasurer Richard Mourdock, a beneficiary of the Tea Party upheaval sweeping the country, defeated him, Mourdock lost the general election to Sen. Joe Donnelly, an Indiana Democrat.
Lugar was also frustrated that Vladimir Putin steadily cut back American funding of Russia's disarmament efforts. Under Putin, CTR came to be seen as a Washington conspiracy to weaken Russia. Putin has now launched a nuclear modernization program that threatens to reverse much of the progress CTR helped achieve. Lugar also told friends that he regretted Washington's lack of priority on reducing and destroying its own nuclear arsenal.
Equally depressing, Lugar lived to see Washington's growing polarization along party and ideological lines, and, as a result, the collapse of bi-partisan efforts like CTR that did so much to protect American national security at a pivotal, dangerous moment. Unlike so many, he never stopped believing that true national security depended on politicians setting aside petty and sometimes profound differences in the nation's interest. We need more public servants like him.


text:
Thomas Villanova isn't the only official from Local 134 of the International Brotherhood of Electrical Workers who participated in a city pension and a union pension at the same time, in violation of state law.
Three other local leaders who did so stand to collect more than $6 million from the municipal pension fund in their lifetimes, according to an analysis by the Tribune and WGN-TV.
Like Villanova, all three retired from the city before the age of 60. Like Villanova, each signed an application saying he wasn't participating in his union's pension fund, and Local 134 officials wrote letters for all of them saying they weren't getting any union pension benefits.
But they were.
Timothy Foley, the union's business manager and financial secretary, retired in 2008 from a $47,000-a-year city job that he hadn't worked at since 1995. That was the year he took a leave of absence from his position as an electrical mechanic to go work for the union.
When he retired from the city, he received credit for the time he worked at the union, and his pension was based on the four-year average of his union salary, which was about $143,000. That allowed him to land a $105,000 city pension, more than double his old city salary, even as he continued to take home more than $160,000 in union pay. He was 54 years old.
Local 134 business agent Michael Nugent took a leave of absence from a $58,000-a-year city job in 2000. When he retired from the city eight years later, at age 54, he began receiving a city pension based on his union salary. The $105,000 he received came on top of his $110,000 union pay.
Municipal pension fund officials discovered in October that Foley and Nugent had received contributions toward their union pensions during a period already covered by their city pensions. Both signed the same affidavit that Villanova did, acknowledging they had been participating in both funds.
Through the local's attorney, Patrick Deady, Foley and Nugent declined to comment. Deady told the Tribune and WGN-TV that the men did what city pension fund officials told them to do.
"They followed the instructions when they initially applied, and they have done everything the pension fund required them to do," Deady said.
Terrance Stefanski, the city pension fund's executive director, acknowledged that the men violated state law by participating in both funds but said they were given the chance to come into compliance because the city fund had no way of determining whether they had done it on purpose.
The first Local 134 leader to receive an inflated city pension was Michael Fedanzo. He retired from a $50,000-a-year city job in 2002 and began collecting a $70,000 city pension while earning about $110,000 from the local as a business agent. He was 55 at the time.
Fedanzo continued to work for the union for another seven years and is entitled to a union pension for that time period. The problem is that he collected union pension money for some years already covered by his city pension, even though he had signed an application stating otherwise.
Peter Cerf, the union official who wrote a letter saying Villanova had waived his right to a union pension, also wrote a similar letter for Fedanzo. (Local 134 President Gary Niederkorn wrote letters for Foley and Nugent.)
Fedanzo said in a telephone interview that he had asked the local in 2002 to take back contributions to his union pension that overlapped with his city pension but that the local failed to do so. Now that the city pension fund has raised the issue, he said he is being forced to pay back $21,000 in pension payments to the union and had his city pension reduced.
"It's really the union who screwed up," he said. "Don't get me wrong, I love the union. But I got screwed by the union."
So far, Fedanzo has received more than $600,000 from his city pension.

domain: news


text:
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domain: agronomy


Jaguar recently unveiled Bike Sense, an experimental technology that alerts a driver of nearby bicycles and motorcycles in an effort to reduce accidents between cars and two-wheeled vehicles. The system uses sensors to identify nearby bikes or motorcycles, and warns the driver with a variety of cues including lights, a bicycle bell sound, and vibrations in the car’s pedals. It also works to reduce “dooring” accidents by flashing a warning light and sending a vibration through the interior door handle if a passenger tries to open the door when a bike is approaching. There’s no word on when the technology might be available in production vehicles.
photo via Jaguar


text: This truck is bringing Mobile BoDillaz (MoBoDillaz) to the RVA area. What's a BoDilla? Well, it's a quesadilla filled with a variety of tasty things, like grilled or breaded chicken, ground beef, tender steak, and a wide range of veggies, sauces, and cheese. If you've ever been by one of the brick-and-mortar BoDillaz you know what to expect, and you know that you're getting great food wherever you bring this truck. If not, stop by and dig in to learn what goes into the making of a famous quesadilla!

domain: food


text:
Best of Finland Russia and The Baltic States
Explore the rare Baltic gems of Lithuania, Estonia, Latvia and Belarus along the seaway from Helsinki to St. Petersburg for fascinating insights into the heritage of the Tsars and days spent seeing the magnificent sights of Moscow. You'll also wander through the ancient capitals of Riga, Vilnius and Minsk and the beautifully preserved streets of Talinn's Haseatic Quarter.
- View Red Square in Moscow, Nevsky Prospekt and St. Isaac's Cathedral in St. Petersburg and the Finlandia Hall in Helsinki
- Visit the St. Peter and Paul Fortress and Hermitage Museum in St. Petersburg, the Kremlin in Moscow, the Peter and Paul Church in Vilnius, the Hill of Crosses in Siauliai and the Alexander Nevsky Cathedral in Tallinn
- 14 hot buffet breakfasts
- 7 dinners (including Be My Guest)
- 1 Regional Meal lunch
- Audio Headsets for flexible sightseeing
- Daily breakfast and an average of 50% of evening meals
- Luxury air-conditioned coach with WiFi in most countries or alternative transportation (such as rail journeys)
- Ferry across the Gulf of Finland.
- Cherry-picked hotels, all tried and trusted
- Hand-picked Insider Experiences
- Optional Experiences and free time
- All porterage and restaurant gratuities
- Must-see sightseeing and surprise extras
- An expert Travel Director and separate Driver
- All hotel tips, charges and local taxes
- High speed train between St. Petersburg and Moscow.
- Rail service is reserved in standard class.
- VIP entry to many sights

domain: travel


text: We all enjoyed a fantastic Active Week here in the Junior School. It was a busy week with plenty of activities for children and parents alike. We made an extra effort to be healthier in every sense by being more active, eating more healthily and thinking positive thoughts. The children went running each day, with some classes even completing a full kilometer each day on a marked out course. We had lots of visitors working with the children on activities such as basketball, tennis, football, Pilates and dancing. We had a parent's fitness class and a parent walk also. Sport's Day was a great success with the children enjoying moving from station to station to join in the fun of badminton, tennis, basketball, uni hoc, football, potato and spoon race, relays, orienteering, parachute activities, sack race, yoga and dance. Thank you to everyone who helped with organising the activities and to those who volunteered their expertise to help make Active Week a varied and fun filled time for all involved.

domain: sociology


How the EE network got even faster
Last year EE, part of BT Group, is the largest and most advanced mobile communications company in the UK teamed up with Sony to achieve the UK’s fastest ever real world mobile download speeds.
Cardiff, London, became the first cities to benefit from increased the 4G network capacity when EE switched on a next generation 4G+ network upgrade. Birmingham, Manchester and Edinburgh followed soon after.
EE’s new 4G+ capability uses cutting edge technology to get even more capacity and faster speeds from existing spectrum resources.
Optimised network performance
At an event in Cardiff city centre, EE demonstrated live download speeds of 429Mbps using Sony’s flagship Xperia XZ Premium smartphone.
Sony’s Xperia XZ Premium is the UK’s first ‘Cat 16’ smartphone optimised for the EE network and EE is the only mobile network able to support the new device’s unique upload and download capabilities. However, all devices on the EE network can benefit from the additional capacity and technology that EE is building into its network.
Peak speeds can be above 400Mbps with the right device, and customers connected to these sites should be able to consistently experience speeds above 50Mbps.
Marc Allera, CEO BT Consumer, said: “Sony has raised the bar in smartphone speeds, and we’re investing and innovating to match that and give our customers the fastest network speeds in the UK.”
He added: “What we demonstrated live in Cardiff is more than ten times the average mobile download speed. We’re rolling out this new capability to the busiest areas of the UK so that our customers always have a great connection to do the things they love.”
How the technology works
The sites that are capable of delivering these maximum speeds are equipped with 30MHz of 1800MHz spectrum, and 35MHz of 2.6GHz spectrum. The 1800MHz carriers are delivered using 4x4 MIMO, which sends and receives four signals instead of just two, making the spectrum up to twice as efficient. The sites also broadcast 4G using 256QAM, or Quadrature Amplitude Modulation, which increases the efficiency of the spectrum.
These two cutting edge technologies applied to EE’s award-winning 4G network help to further improve customer experience by increasing capacity, meaning faster speeds and a more reliable data experience.
EE runs the UK's biggest and fastest mobile network, having pioneered the UK's first superfast 4G mobile service in October 2012 and was the first European operator to surpass 14 million 4G customers in December 2015.


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The Gallop organization, a research firm based in the USA, asked citizens of 142 countries about their confidence in local policing, feelings of safety while walking alone and personal experiences of crime.
Gallup interviewed more than 148,000 people for the 2018 report. Gallup’s rankings are based on residents’ own sense of security.
2. (tied) Finland, Iceland, and Norway
5. (tied) Hong Kong and Uzbekistan
7. (tied) Canada and Switzerland
10. (tied) Denmark, Slovenia, Luxembourg, Austria, China, Netherlands, Egypt
More at CNN
Eva Holland, writing for Outside Magazine:
Students in the Extreme Polar Training course, a two-week freeze-fest held near the Arctic Circle on Canada’s Baffin Island, learn how to live in Earth’s coldest conditions. Still, nothing really prepares you for 72 hours of a sled-pulling, pathfinding ordeal on a skinny pair of skis.
As I slogged through deep snow and deeper darkness toward my tent, tripping and scraping my shins on chunks of broken ice concealed by fresh powder, I reminded myself that I had come here intending to suffer.
As I settled in for sleep, I felt hope for the first time that there might be more to polar travel than fear and misery. But the night still seemed deadly: The tent thrashed around in a heavy wind, the huskies chained nearby howled, and the ice creaked and boomed as it shifted in the grip of the tide.
I tried to focus on the harsh beauty of the ice walls lining the frozen channel we were moving through. I was determined to bury my fears in sheer wonder and suffocate them.
Katie O’Reilly, writing for Sierra Magazine:
First things first: Make sure your base-layer game is strong.
“What most people don’t realize is that when it’s 30 to 40 degrees below zero, the concern isn’t about getting too cold, but rather getting too hot,” Larsen cautions. “When you sweat, moisture builds up and can replace the layer of air next to your skin.” Hence, the importance of a close-fitting base layer made from moisture-wicking fabric that allows for ventilation. Larsen prefers the wide range of base layers available through Norwegian technical outfitter Helly Hansen. “They make a variety, and I layer up and down depending on the temperature and the activity. For polar expeditions, they have a great line of mountain apparel.”
Get down with down.
Larsen describes a big down jacket as his “last defense against really cold weather—whether you’re in a sub-zero-degree tent or just walking around town.” He explains, “It’s the lightest-weight and warmest of insulators,” and says he relies on Helly Hansen’s shell jackets. “They use Allied Feather & Down, which is sustainably harvested.”

domain: geography


text:
21 Porcelain: surface stories with Mollie Bosworth
Ceramic artist – North QLD
Mollie's work is driven by materials and processes and influence by textures and patterns in nature, often drawn from her love of gardening and the tropical North Queensland environment in which she lives.
Course Fee (Adult): $745
Course Fee (Child): $
Course Material Fee: $70
Course Date/s:03-Jan-2019 07-Jan-2019
Current Capacity: Full
You can register for this course online. Just fill in the details below, supply your payment details and our course coordinator will secure your position.
You will need to complete this form for each person attending.

domain: artistic


NASA is getting ready to launch deep-space capsule Orion next month on its first space flight. A NASA administrator lately called this mission ‘our first step in our journey to Mars’.
Deputy Associate Administrator William Hill and other NASA and industry officials have expressed high hope from the mission. Orion is expected to launch on December 4. The mission will blast an unmanned Orion capsule from Kennedy Space Center, Fla., sending it over 3,500 miles into space and back for a splashdown off Baja California, Mexico.
The flight, which will include two Earth orbits and last less than five hours, will provide NASA and its Orion business partner Lockheed Martin their first space test of the capsule that imagined as a vital part of any NASA trips to the moon, an asteroid, Mars or beyond.
For the test on December 4, Orion will be staged on top of the most powerful rocket accessible in the world today, a three-booster Delta IV Heavy, given by United Space Alliance. As launched, the capsule will be completely configured to take four crew members, although it will be unoccupied.
NASA will be performing all the tests and research on the flight with the assumption that there are astronauts on board. The launch is scheduled for 7:05 a.m., with December 5 and 6 available as backup launch days.
The mission will test Orion’s capabilities ranging from the 17 separations that will take place as many parts of the rocket. The test flight will cost about $370 million including the rocket and it will not include the capsule, which NASA and Lockheed Martin plan to recover and use again.
“This mission is significant in that it enables human space flight to deep space and to destinations that we have yet to imagine .We intend to test ourselves on this mission, and we intend to test our spacecraft”, said NASA’s Orion Flight Director Mike Sarafin.


text:
Key Benefits of Chronos®
Chronos® is leading the way in chemical free and ultrafast automated high level disinfection for reprocessing ultrasound probes.
When seconds count, Chronos® delivers a
solution that reduces cross-infection risk, for seamless Ultrasound High Level Disinfection (HLD)
Chronos® ultrafast 90 seconds HLD cycle time, streamlines clinical workflows to drastically reduce time and costs
Chronos faster clinical workflows are extremely efficient in quickly disinfecting ultrasound probes between patients to maximise availability of probes, for staff and patient examinations.1
“Speed, simplicity, efficacy. It means everything! Thanks to this speed process I multiplied by 5 the number of vaginal ultrasounds performed daily. I am extremely satisfied by the system. I have waited a long time for a solution of this kind, today I could not work without it!”
Dr. Clabeau, Gynecologist European Hospital Georges Pompidou
Chronos® Streamlines Clinical Workflows: Cleaning and Disinfecting ultrasound probes between patients within 101secs. 4
While other HLD Systems can Take Significant Longer in Cleaning, Disinfecting and Rinsing/Drying, with multiple glove changes, between patients.1,4
Chrono’s Automated Verification System replaces the manual process of checking chemical indicators and test strips.
- This breakthrough technology consists of a monitoring and an independent control system.
The monitoring system consists of two photodiodes that continually measure the UV-C dose to operate the disinfection cycle to ensure High Level Disinfection efficacy is achieved. This takes approximately 90 seconds.
An Independent control system validates the disinfection cycle based on the UV-C dose measurements of a third photodiode, declaring the disinfection cycle completed “CYCLE OK”.
The Automated verification system operates similar to Parametric Release
- providing extra reassurance and saves time in automatically validating every disinfection cycle.
Chronos® simple “one button” process to activate HLD improves compliance and audit readiness
Fully automated with one push button process versus multiple steps ensures a reproducible disinfection cycle every time. Thus, reduces operator error and improves compliance
- Simple plug and play design that minimising staff interaction and training requirements
- All that is needed is an electrical connection as there are NO other running costs
- No added cost in purchasing, storing, and handling consumables.
- Disinfects probe, handle and cable section
- Chronos is mobile within departments which is ideal for POCUS to support quick diagnosis and management
Traceability for Ultrasound Disinfection
Chronos traceability system electronically captures and stores data on Chronos during the disinfection cycle.
Germitrac® is an interface that visually displays all disinfection records through a web browser
Data can be easily accessed via a computer or hospital network linked to the Chronos.
Provides users with numerous functionalities:
- Disinfection Cycles – View and Filter Data
- Disinfection Comments Field
- Print and download report
- View tickets
Integrated – with the hospital network
Automated through a third-party software accessing Germitrac API web services to retrieve disinfection data and link it to a patient record, for example Electronic Medical Records (EMR) or Electronic Patient Record (EPR)
Chronos® is pioneering UV-C High Level Disinfection (HLD) as the first chemical free system. It has been proven to be effective against an extended range of clinically pathogens, including native human papillomavirus (HPV), providing extra patient safety in reducing cross-infection risks.2,3
Chronos technology works by delivering UV-C high level disinfection (HLD) as light particles and waves reflected from the chamber walls ensures that there is proper illumination and efficient exposure on the probe surfaces
UV-C HLD energy of the photons penetrates the cell walls of microorganisms (bacteria, fungi, viruses, mycobacteria and spores), which is absorbed by DNA, RNA and the proteins leaving them unable to perform cellular functions and replication therefore destroying them
Independent Microbicidal Efficacy
Chronos microbial efficacy has been independently tested and complies with testing requirements set by different regulators.
Chronos is the only system proven to kill high-risk HPV both in vitro and in clinical use.2-3
Non-Corrosive and Compatible with Your Ultrasound Probes
Chronos is compatible with many leading ultrasound probe manufacturers, supported by extensive probe compatibility testing.
“It is safer for patients because we do not use corrosive chemicals anymore…
It is the less corrosive HLD technique. Before UV HLD, our probe’s shelf life was very short. Last year for example we had to buy 4 or 5 vaginal ultrasound probes. Since UV, we practically use one probe only.”
Pierre Leger, Head of Biomedical Department and Edith Gumbeau, nurse, Foundation St. Joseph, Marseille
An easy probe loading and unloading mechanism reduces wear and tear on probe cable caused by cable clamps.
Safeguards staff and patients by avoiding hazardous chemicals, fumes, and spills.
No chemical residuals are left on the probe after disinfection, protecting the operator and patient from chemical burns (hand or face).
Providing an environmentally friendly examination room that is ideal for point of care ultrasound (POCUS).
Chronos® Brochure, Quick Reference Guide and User Manuals
Download the Hypernova Chronos Product Brochure, Accessories Brochure and Probe Compatibility Fact Sheet
An extensive summary of all the benefits Chronos Offers in 90 seconds
Johnson et al., Evaluation of a Hydrogen Peroxide-Based System for High-Level Disinfection of Vaginal Ultrasound Probes, Journal of Ultrasound Medicine, 32:1799-804, 2013.
Meyers J, Ryndock E, Conway MJ, Meyers C, Robison R. Susceptibility of high-risk human papillomavirus type 16 to clinical disinfectants. J Antimicrob Chemother. 2014;69(6):1546-50
Meyers C, Milici J, Robison R (2017) UVC radiation as an effective disinfectant method to inactivate human papillomaviruses. PLoS ONE 12 (10): e0187377.


Insituformâs Mack Meher checks the water flow that fills the white resin impregnated fabric lining that goes down the manhole here, and through the existing 50 year old sanitary sewer pipe, to the next manhole, on Tuesday, July 19, 2011; part of Buena Vista Township Phase II Sewer Project, in Michigan, where the United States Department of Agriculture funded the refurbishment and rehabilitation of 45,500 linear feet of defective sewer pipe, and more than 200 manholes; on Tuesday, July 19, 2011. The project includes the construction of an off-site excess flow basin; construction of a pump station; and improvements at the wastewater treatment plant. The system will serve 2,349 residential and 177 commercial customers and will bring the township into compliance with environmental regulations regarding pollutant discharges.
Inspections revealed that the 1960s sanitary sewer system had inflow from storm and rainwater systems and overflowed into the sanitary sewer system. Infiltration was found in the form of cracks, and other failures that allowed groundwater to enter. Rather than digging up the old system, rehabilitation with a cured-in-place-pipe (CIPP) method was chosen. This provides up to a 50% savings and the liner is projected to last 50-100 years. CIPP repair of the 8-30â diameter pipes involves pulling a resin-saturated fabric liner through a damaged pipe, which is then cured with steam or hot water to form a tight-fitting, joint less replacement pipe. Trenchless repair methods require less digging than traditional âdig and replaceâ repair methods and minimize damage to yards and landscaping. There are two of these teams and one manhole rehabilitation to seal the underground casting frame or brick structure. They process 600-800 feet per day.
Wade Trim Inc. Professional Engineer Tiffany Harrison said about working with the United States Department of Agricultureâs Rural Development âIt was very easy, they make it very straight forward, in what paperwork they need. They were always available for questions.
USDA Rural Development has funded water and sewer projects in 117 Michigan communities serving more than 94,000 customers.
Senior Advisor to the Secretary of Agriculture Doug OâBrien said, âAmerica will win the future by out-building, out-educating and out-innovating the world,â O'Brien said. âThe Obama Administration is committed to the residents and business owners of Buena Vista Township in building the infrastructure needed to protect our environment and ensure clean water for future generations.â Being almost entirely surrounded by the Great Lakes, Michigan residents know that a small problem upstream usually leads to much bigger problems downstream and that the impact of water pollution extends far beyond the immediate community.
USDA photo by Lance Cheung.
May 11, 2014


More than 95 million people in 20 different states around the country were in a state of emergency due to the tornadoes on April 12. There were 60 reports of tornadoes and at least 33 people were killed in the nine southern states that were hit.
A dozen tornadoes were reported in Texas during Easter. The first tornado was reported in Fredericksburg around 4.30 AM. This storm grew weaker though, as it moved from northeastern Gillespie into northwestern Blanco County. Storms continued hitting hard and around 5:30 AM, another tornado was reported by the National Weather Service.
Over a million houses, buildings, and structures were damaged due to the storms. There are about 103,002 people without power in the south due to the tornadoes, and 758,657 of those customers are in Texas according to PowerOutage.us, a website that tracks power outages in the United States. Utility companies are still working on restoring power in Texas.
“Estimates from Blanco County Emergency Management show that a total of approximately 60 residences were, with at least eleven being completely destroyed and eight more sustaining major damage. There were two minor injuries,” National Weather Service said.
In South Carolina, nine people were killed as a result of the storms. The tornadoes caused severe damage in the affected area, including damaging utilities. The Hampton County Emergency Management Agency reported that at least five of the deaths came from their county.
“In Nixville and Estill, we’re looking at serious devastation in those areas,” South Carolina Hampton County Council Chairman Clay Bishop said to the Post and Courier. “There’s [sic] numerous houses destroyed.”
At least seven people were killed, twelve were hurt, and two were reported missing on April 13 in Georgia. 20 people were injured and more than 150 structures were damaged or destroyed in Tennessee.
“We’re still in the rescue mode and that’s pretty much going through the area which was affected, going door to door to make sure that we got everyone accounted for,” Hamilton County Emergency Management Communications Director Amy Maxwell said. “Obviously, there’s significant damage in East Brainerd and East Ridge, but the total destruction is in East Ridge.”
The tornadoes have caused a lot of damage, but emergency services are working to repair the damage. In order to stay safe, professionals recommend having your phone on the whole day. This will ensure you are able to receive notifications on any dangerous weather.


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For one night, Maysville will be Hollywood along the Ohio River, thanks to George Clooney.
The 46-year-old actor-director will visit the northern Kentucky town Monday night for a special screening of his new movie, “Leatherheads,” his father, Nick Clooney, said Tuesday.
“The whole town is abuzz,” said Maysville Mayor David Cartmell.
Clooney, who won an Oscar for his role in “Syriana,” grew up in the town of Augusta, about 16 miles west of Maysville.
The setting has special significance for the Clooney family. In 1953, “The Stars Are Singing,” starring Rosemary Clooney, George Clooney’s aunt, premiered in Maysville.
“He knows all about that, and he is very interested in kind of connecting the dots as far as family history,” Nick Clooney said. “He thinks that would be kind of cool for the family, and one hopes for the town, too.”
Nick and Rosemary Clooney were born in Maysville. George Clooney was born in Lexington, and the family moved several times before settling in Augusta.
The movie will be shown at the Washington Opera Theatre, Nick Clooney and Universal Studios said Tuesday.
Cartmell said Monday night’s screening will probably be the biggest event in the town of about 9,000 since the premiere of “The Stars Are Singing.”
It will also be the hottest ticket in town.
Nick Clooney said about 300 seats will be available. The opera house seats about 450 people, but equipment needed to show the movie will take up space in the balcony, he said.
Cartmell said he expects the town to catch Hollywood fever.
“Next week, we’ll be Hollywood on the Ohio,” he said.