The present invention relates to a method for producing cancerous disease modifying antibodies using a novel paradigm of screening. By segregating the anti-cancer antibodies using cancer cell cytotoxicity as an end point, the process makes possible the production of anti-cancer antibodies for therapeutic and diagnostic purposes. The antibodies can be used in aid of staging and diagnosis of a cancer, and can be used to treat primary tumors and tumor metastases. The anti-cancer antibodies can be conjugated to toxins, enzymes, radioactive compounds, cytokines, interferons, target or reporter moieties and hematogenous cells.

TROP-2 is a cell surface glycoprotein expressed on most carcinomas, as well as some normal human tissues. It was initially defined as a molecule recognized by two murine monoclonal antibodies raised to a human choriocarcinoma cell line BeWo that recognized an antigen on human trophoblast cells (Faulk 1978). The same molecule was independently discovered by other investigators which led to multiple names describing the same antigen. Hence, TROP-2 was also referred to as GA733-1 and epithelial glycoprotein-1 (EGP-1) (Basu 1995, Fornaro 1995).
The TROP-2 gene is an intronless gene that was thought to have been formed through the retroposition of a homologous gene GA 733-2 (also known as epithelial glycoprotein-2, EpCAM and Trop-1) via an RNA intermediate. The TROP-2 gene has been mapped to chromosome 1p32 (Calabrese 2001). The protein component of TROP-2 has a molecular mass of approximately 35 kilodaltons. Its mass may be increased by 11-13 kilodaltons with heterogeneous N-linked glycosylation of its extracellular domain. There are many cysteine residues in the extracellular domain which could form disulfide bridge sites. TROP-2 is a substrate for protein kinase C, a Ca2+ dependent protein kinase and the intracellular serine 303 residue has been shown to be phosphorylated (Basu 1995). It has also been shown that crossing-linking of TROP-2 with anti-TROP-2 antibodies transduced a calcium signal as shown by a rise in cytoplasmic Ca2+ (Ripani 1998). These data support signal transduction as a physiological function of TROP-2, although to date no physiological ligand has been identified. Recently an association between TROP-2 expression and cancer has been shown as TROP-2 was identified as a member of a group of genes reported to be the most highly overexpressed in ovarian serous papillary carcinoma compared to normal ovarian epithelium in a large-scale gene expression analysis using cDNA microarray technology (Santin 2004).
The expression profile of TROP-2 has been elucidated through immunohistochemistry (IHC) and flow cytometery studies using many different TROP-2 antibodies. Anti-TROP-2 antibodies 162-25.3 and 162-46.2 were produced through immunization of mice with the human choriocarcinoma cell line BeWo, and were investigated for their reactivity to a series of tumor and lymphoid cell lines and peripheral blood mononuclear cells. In this study both antibodies appeared to be trophoblast specific, staining 3 of the 4 choriocarcinoma cell lines tested, while none of the other lymphoid or tumor cell lines (representing fibrosarcoma, cervical sarcoma, colon carcinoma, melanoma, neuroblastoma, erythroleukemia) were stained in an indirect immunofluorescence FACS assay. In addition, none of the normal peripheral blood cells were stained. The antibodies were tested for staining of formalin-fixed paraffin-embedded placenta tissue sections and frozen normal sections of liver, kidney, spleen, thymus and lymph node tissues. The placenta tissue sections were stained with both antibodies, while there was no staining of the other normal tissues (Lipinski 1981). These two antibodies have strictly been reported for use in in vitro diagnostic studies.
Anti-TROP-2 antibody MOv16 was generated through the immunization of mice with a crude membrane preparation of poorly differentiated ovarian carcinoma OvCa4343/83. MOv16 was tested for reactivity to a series of frozen tissue sections of benign and malignant ovarian tumors. MOv16 reacted with 31 of 54 malignant ovarian tumors and 2 of 16 benign ovarian tumors. Of the 5 mucinous ovarian tumors that were tested, MOv16 was completely unreactive. MOv16 was also tested for reactivity to frozen sections of non-ovarian malignant tumors where it was found to bind 117 of 189 breast carcinoma sections and 12 of 18 lung carcinoma sections. MOv16 was completely unreactive on 16 non-epithelial tumors that were tested (including liposarcomas, chondrosarcomas, endotheliomas, histiocytomas and dysgerminomas). When tested on frozen normal tissue sections, MOv-16 was reactive with breast, pancreas, kidney and prostate sections. MOv16 reactivity was reported to be negative on lung, spleen, skin, ovary, thyroid, parotid gland, stomach, larynx, uterus and colon sections, though the number of tissue sections that were used was not reported. The authors noted that frozen tissue sections were used because MOv16 was unreactive to paraffin embedded tissues (Miotti 1987). This antibody has also only been reported for use in in vitro diagnositic studies.
Anti-TROP-2 antibody Rs7-3G11 (RS7) was generated through the immunization of mice with a crude membrane preparation derived from a surgically removed human primary squamous cell carcinoma of the lung. IHC was used to examine the staining of RS7 on frozen sections of human tumor and normal tissues. RS7 bound to 33 of the 40 sections representing tumors of the breast, colon, kidney, lung, prostate and squamous cell cancer. Of the normal tissues RS7 bound to 16 of 20 sections of breast, colon, kidney, liver, lung and prostate tissues while none of the five sections of spleen tissue were stained. In this study the authors noted that it appeared that antigen density in tumors was higher than in normal epithelial tissues (Stein 1990).
Additional studies of the tissue specificity of RS7 were carried out on both tumor and normal tissues. RS7 was tested on a panel of frozen tumor sections and bound to 65 of the 77 sections representing tumors of the lung, stomach, kidney, bladder, colon, breast, ovary, uterus and prostate. There was no binding to the 5 lymphomas tested. RS7 was tested on a panel of 85 frozen human normal tissue sections composed of a total of 24 tissue types. 39 sections of 13 normal tissues (lung, bronchus, trachea, esophagus, colon, liver, pancreas, kidney, bladder, skin, thyroid, breast and prostate) were stained by RS7. The authors of this study noted that in the tissues in which positive staining was observed, the reactivity was generally restricted to epithelial cells, primarily in ducts or glands. It was also noted that this study was limited to frozen sections since it was observed that RS7 was not reactive on formalin-fixed paraffin-embedded sections (Stein 1993).
Polyclonal anti-TROP-2 antibodies were prepared by immunizing mice with a synthetic peptide corresponding to amino acid positions between 169 and 182 of the cytoplasmic domain of human TROP-2. The polyclonal antibodies were tested on a tissue array slide that contained formalin-fixed human esophageal hyperplasia and carcinoma tissues. Ten of the 55 carcinoma specimens displayed heavy staining with the polyclonal antibodies, while the mild hyperplasia tissue stained very weakly, indicating expression levels may be related to malignant transformation (Nakashima 2004).
Overall, IHC reactivity patterns obtained with different anti-TROP-2 antibodies were consistent. Expression in cancer was seen primarily in carcinomas, and most carcinomas were reactive. In normal tissues, expression appeared to be limited to cells of epithelial origin, and there was some evidence that staining of carcinomas was stronger than staining of corresponding normal epithelial tissues.
In addition to being used in IHC studies, antibody RS7 was tested in in vivo models with initial experiments consisting of tumor targeting studies in nude mouse xenograft models. Radiolabeled RS7 injected i.v. was shown to accumulate specifically in the tumor of mice bearing either Calu-3 (lung adenocarcinoma) or GW-39 (colon carcinoma) tumors (Stein 1990). Further studies were done to investigate the biodistribution of radiolabeled RS7 in a xenograft system and to study the therapeutic potential of RS7 as an immunoconjugate. In this study the therapeutic efficacy of 131I-labeled RS7 F(ab′)2 was investigated in nude mice bearing Calu-3 human lung adenocarcinoma xenografts. Three weeks following inoculation of the mice with Calu-3 cells, when the tumors had reached a size of approximately 0.3-0.9 grams, groups of 6-7 mice were treated with a single dose i.v. of either 1.0 mCi 131I-RS7-F(ab′)2 or 1.5 mCi 131I-RS7-F(ab′)2 and compared to a similar group of untreated control mice. The single dose of 1.0 mCi 131I-RS7-F(ab′)2 resulted in tumor growth suppression for approximately 5 weeks, while the single dose of 1.5 mCi 131I-RS7-F(ab′)2 resulted in tumor regression, and the mean tumor size did not exceed the pre-therapy size until the eighth week after radioantibody injection. Mice receiving the 1.5 mCi 131I-RS7-F(ab′)2 dose experienced a mean body weight loss of 18.7 percent, indicating there was toxicity associated with the treatment. In this study, effects of treatment with naked RS7 or the F(ab′)2 fragment of RS7 were not tested (Stein 1994a). Another study was done to test the efficacy of 131I-RS7 in a MDA-MB-468 breast cancer xenograft model. Groups of ten mice bearing MDA-MB-468 tumors of approximately 0.1 cm3 were treated with a single dose i.v. of either 250 microcuries 131I-RS7 or 250 microcuries 131I—Ag8 (an isotype matched control antibody). Groups of six mice were treated with a single dose i.v. of 30 micrograms of either unlabeled RS7 or Ag8. Complete regression of the tumors (except for one animal that had a transient reappearance of tumor) was seen in the animals treated with 131I-RS7, which lasted for the duration of the 11 week observation period. Tumor regression was also seen in 131I-Ag8 treated mice, though was only observed between 2 weeks and 5 weeks with tumors either persisting or continuing to grow for the remainder of the study. Tumor growth of mice that received unlabeled RS7 or Ag8 was not inhibited and there did not appear to be any differences in the mean tumor volume of RS7 treated mice compared to the Ag8 treated mice. Two additional groups of 10 mice bearing larger MDA-MB-468 tumors of approximately 0.2-0.3 cm3 were treated with a slightly higher single dose of either 275 microcuries 131I-Rs7 or 275 microcuries 131Ag8 and compared to a similar group of untreated mice. Tumor volume was measured weekly for 15 weeks. Although in this case there was a significant difference in tumor growth between the 131I-RS7 treated mice compared to the untreated mice, there was no significant difference in the tumor growth of the 131I-RS7 compared to the 131I—Ag8 treated mice, indicating a portion of the efficacy may have been due to non-specific effects of the radiation. Unlabeled antibodies were not tested in mice containing 0.2-0.3 cm3 tumors (Shih 1995).
There have been numerous additional studies examining the efficacy of RS7 as an immunoconjugate with an attempt to select the optimal radiolabel for radioimmunotherapy (Stein 2001a, Stein 2001b, Stein 2003). A humanized version of RS7 has also been generated, however it has only been tested in preclinical xenograft models as a radioconjugate (Govindan 2004). These studies show similar positive effects as the previously described studies with RS7, however in one study, even when radiolabeled RS7 was delivered at a previously determined maximum tolerable dose, toxicity occurred leading to death in some mice (Stein 2001a). Although effective treatment of xenograft tumors in mice was achieved with radiolabeled RS7 in these studies, naked RS7 was not evaluated.
Immunizing mice with neuramindase pre-treated H3922 human breast carcinoma cells produced the anti-TROP-2 monoclonal antibody BR110 (as disclosed in U.S. Pat. No. 5,850,854, refer to Prior Patents section). By immunohistology, using human frozen tissue specimens, BR110 was shown to react with a wide range of human carcinoma specimens including those of the lung, colon, breast, ovarian, kidney, esophagus, pancreas, skin, lung and tonsil. No human normal tissue sections were tested. In vitro studies demonstrated that BR110 had no ADCC or CDC activity on the human carcinoma cell lines H3396 or H3922. In vitro studies analyzing the cytotoxicity of BR110-immunotoxins was performed on the human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981. The EC50 for the cell lines tested was 0.06, 0.001, 0.05, 0.09 and >5 micrograms/mL respectively. No cytotoxicity data was disclosed for the naked BR110 antibody. No in vivo data was disclosed for the naked or immunoconjugated BR110.
A number of additional antibodies have been generated that target TROP-2, such as MR54, MR6 and MR23 which were generated from immunization of mice with the ovarian cancer cell line Colo 316 (Stein 1994b) and antibody T16 which was generated by immunization of mice with the T24 bladder cancer cell line (Fradet 1984). The use of these antibodies has been limited to biochemical characterization of the TROP-2 antigen and cell line and tissue expression studies. There have been no reports of anti-cancer efficacy of these antibodies, either in vitro or in vivo. RS7 was the only antibody that was tested for therapeutic efficacy in preclinical cancer models, with its use being limited to a carrier of radioisotope. There are no reports of any naked TROP-2 antibodies exhibiting therapeutic efficacy in preclinical cancer models either in vitro or in vivo.
Monoclonal Antibodies as Cancer Therapy: Each individual who presents with cancer is unique and has a cancer that is as different from other cancers as that person's identity. Despite this, current therapy treats all patients with the same type of cancer, at the same stage, in the same way. At least 30 percent of these patients will fail the first line therapy, thus leading to further rounds of treatment and the increased probability of treatment failure, metastases, and ultimately, death. A superior approach to treatment would be the customization of therapy for the particular individual. The only current therapy which lends itself to customization is surgery. Chemotherapy and radiation treatment cannot be tailored to the patient, and surgery by itself, in most cases is inadequate for producing cures.
With the advent of monoclonal antibodies, the possibility of developing methods for customized therapy became more realistic since each antibody can be directed to a single epitope. Furthermore, it is possible to produce a combination of antibodies that are directed to the constellation of epitopes that uniquely define a particular individual's tumor.
Having recognized that a significant difference between cancerous and normal cells is that cancerous cells contain antigens that are specific to transformed cells, the scientific community has long held that monoclonal antibodies can be designed to specifically target transformed cells by binding specifically to these cancer antigens; thus giving rise to the belief that monoclonal antibodies can serve as "Magic Bullets" to eliminate cancer cells. However, it is now widely recognized that no single monoclonal antibody can serve in all instances of cancer, and that monoclonal antibodies can be deployed, as a class, as targeted cancer treatments. Monoclonal antibodies isolated in accordance with the teachings of the instantly disclosed invention have been shown to modify the cancerous disease process in a manner which is beneficial to the patient, for example by reducing the tumor burden, and will variously be referred to herein as cancerous disease modifying antibodies (CDMAB) or "anti-cancer" antibodies.
At the present time, the cancer patient usually has few options of treatment. The regimented approach to cancer therapy has produced improvements in global survival and morbidity rates. However, to the particular individual, these improved statistics do not necessarily correlate with an improvement in their personal situation.
Thus, if a methodology was put forth which enabled the practitioner to treat each tumor independently of other patients in the same cohort, this would permit the unique approach of tailoring therapy to just that one person. Such a course of therapy would, ideally, increase the rate of cures, and produce better outcomes, thereby satisfying a long-felt need.
Historically, the use of polyclonal antibodies has been used with limited success in the treatment of human cancers. Lymphomas and leukemias have been treated with human plasma, but there were few prolonged remission or responses. Furthermore, there was a lack of reproducibility and there was no additional benefit compared to chemotherapy. Solid tumors such as breast cancers, melanomas and renal cell carcinomas have also been treated with human blood, chimpanzee serum, human plasma and horse serum with correspondingly unpredictable and ineffective results.
There have been many clinical trials of monoclonal antibodies for solid tumors. In the 1980s there were at least four clinical trials for human breast cancer which produced only one responder from at least 47 patients using antibodies against specific antigens or based on tissue selectivity. It was not until 1998 that there was a successful clinical trial using a humanized anti-Her2/neu antibody (Herceptin®) in combination with CISPLATIN. In this trial 37 patients were assessed for responses of which about a quarter had a partial response rate and an additional quarter had minor or stable disease progression. The median time to progression among the responders was 8.4 months with median response duration of 5.3 months.
Herceptin® was approved in 1998 for first line use in combination with Taxol®. Clinical study results showed an increase in the median time to disease progression for those who received antibody therapy plus Taxol® (6.9 months) in comparison to the group that received Taxol® alone (3.0 months). There was also a slight increase in median survival; 22 versus 18 months for the Herceptin® plus Taxol® treatment arm versus the Taxol® treatment alone arm. In addition, there was an increase in the number of both complete (8 versus 2 percent) and partial responders (34 versus 15 percent) in the antibody plus Taxol® combination group in comparison to Taxol® alone. However, treatment with Herceptin® and Taxol® led to a higher incidence of cardiotoxicity in comparison to Taxol® treatment alone (13 versus 1 percent respectively). Also, Herceptin® therapy was only effective for patients who over express (as determined through immunohistochemistry (IHC) analysis) the human epidermal growth factor receptor 2 (Her2/neu), a receptor, which currently has no known function or biologically important ligand; approximately 25 percent of patients who have metastatic breast cancer. Therefore, there is still a large unmet need for patients with breast cancer. Even those who can benefit from Herceptin® treatment would still require chemotherapy and consequently would still have to deal with, at least to some degree, the side effects of this kind of treatment.
The clinical trials investigating colorectal cancer involve antibodies against both glycoprotein and glycolipid targets. Antibodies such as 17-1 A, which has some specificity for adenocarcinomas, has undergone Phase 2 clinical trials in over 60 patients with only 1 patient having a partial response. In other trials, use of 17-1 A produced only 1 complete response and 2 minor responses among 52 patients in protocols using additional cyclophosphamide. To date, Phase III clinical trials of 17-1A have not demonstrated improved efficacy as adjuvant therapy for stage III colon cancer. The use of a humanized murine monoclonal antibody initially approved for imaging also did not produce tumor regression.
Only recently have there been any positive results from colorectal cancer clinical studies with the use of monoclonal antibodies. In 2004, ERBITUX® was approved for the second line treatment of patients with EGFR-expressing metastatic colorectal cancer who are refractory to irinotecan-based chemotherapy. Results from both a two-arm Phase II clinical study and a single arm study showed that ERBITUX® in combination with irinotecan had a response rate of 23 and 15 percent respectively with a median time to disease progression of 4.1 and 6.5 months respectively. Results from the same two-arm Phase II clinical study and another single arm study showed that treatment with ERBITUX® alone resulted in an 11 and 9 percent response rate respectively with a median time to disease progression of 1.5 and 4.2 months respectively.
Consequently in both Switzerland and the United States, ERBITUX® treatment in combination with irinotecan, and in the United States, ERBITUX® treatment alone, has been approved as a second line treatment of colon cancer patients who have failed first line irinotecan therapy. Therefore, like Herceptin®, treatment in Switzerland is only approved as a combination of monoclonal antibody and chemotherapy. In addition, treatment in both Switzerland and the US is only approved, for patients as a second line therapy. Also, in 2004, AVASTIN® was approved for use in combination with intravenous 5-fluorouracil-based chemotherapy as a first line treatment of metastatic colorectal cancer. Phase III clinical study results demonstrated a prolongation in the median survival of patients treated with AVASTIN® plus 5-fluorouracil compared to patients treated with 5-fluourouracil alone (20 months versus 16 months respectively). However, again like Herceptin® and ERBITUX®, treatment is only approved as a combination of monoclonal antibody and chemotherapy.
There also continues to be poor results for lung, brain, ovarian, pancreatic, prostate, and stomach cancer. The most promising recent results for non-small cell lung cancer came from a Phase II clinical trial where treatment involved a monoclonal antibody (SGN-15; dox-BR96, anti-Sialyl-LeX) conjugated to the cell-killing drug doxorubicin in combination with the chemotherapeutic agent TAXOTERE®. TAXOTERE® is the only FDA approved chemotherapy for the second line treatment of lung cancer. Initial data indicate an improved overall survival compared to TAXOTERE® alone. Out of the 62 patients who were recruited for the study, two-thirds received SGN-15 in combination with TAXOTERE® while the remaining one-third received TAXOTERE® alone. For the patients receiving SGN-15 in combination with TAXOTERE®, median overall survival was 7.3 months in comparison to 5.9 months for patients receiving TAXOTERE® alone. Overall survival at 1 year and 18 months was 29 and 18 percent respectively for patients receiving SNG-15 plus TAXOTERE® compared to 24 and 8 percent respectively for patients receiving TAXOTERE® alone. Further clinical trials are planned.
Preclinically, there has been some limited success in the use of monoclonal antibodies for melanoma. Very few of these antibodies have reached clinical trials and to date none have been approved or demonstrated favorable results in Phase III clinical trials.
The discovery of new drugs to treat disease is hindered by the lack of identification of relevant targets among the products of 30,000 known genes that could contribute to disease pathogenesis. In oncology research, potential drug targets are often selected simply due to the fact that they are over-expressed in tumor cells. Targets thus identified are then screened for interaction with a multitude of compounds. In the case of potential antibody therapies, these candidate compounds are usually derived from traditional methods of monoclonal antibody generation according to the fundamental principles laid down by Kohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein). Spleen cells are collected from mice immunized with antigen (e.g. whole cells, cell fractions, purified antigen) and fused with immortalized hybridoma partners. The resulting hybridomas are screened and selected for secretion of antibodies which bind most avidly to the target. Many therapeutic and diagnostic antibodies directed against cancer cells, including Herceptin® and RITUXIMAB, have been produced using these methods and selected on the basis of their affinity. The flaws in this strategy are two-fold. Firstly, the choice of appropriate targets for therapeutic or diagnostic antibody binding is limited by the paucity of knowledge surrounding tissue specific carcinogenic processes and the resulting simplistic methods, such as selection by overexpression, by which these targets are identified. Secondly, the assumption that the drug molecule that binds to the receptor with the greatest affinity usually has the highest probability for initiating or inhibiting a signal may not always be the case.
Despite some progress with the treatment of breast and colon cancer, the identification and development of efficacious antibody therapies, either as single agents or co-treatments, have been inadequate for all types of cancer.
Prior Patents:
U.S. Pat. No. 5,750,102 discloses a process wherein cells from a patient's tumor are transfected with MHC genes which may be cloned from cells or tissue from the patient. These transfected cells are then used to vaccinate the patient.
U.S. Pat. No. 4,861,581 discloses a process comprising the steps of obtaining monoclonal antibodies that are specific to an internal cellular component of neoplastic and normal cells of the mammal but not to external components, labeling the monoclonal antibody, contacting the labeled antibody with tissue of a mammal that has received therapy to kill neoplastic cells, and determining the effectiveness of therapy by measuring the binding of the labeled antibody to the internal cellular component of the degenerating neoplastic cells. In preparing antibodies directed to human intracellular antigens, the patentee recognizes that malignant cells represent a convenient source of such antigens.
U.S. Pat. No. 5,171,665 provides a novel antibody and method for its production. Specifically, the patent teaches formation of a monoclonal antibody which has the property of binding strongly to a protein antigen associated with human tumors, e.g. those of the colon and lung, while binding to normal cells to a much lesser degree.
U.S. Pat. No. 5,484,596 provides a method of cancer therapy comprising surgically removing tumor tissue from a human cancer patient, treating the tumor tissue to obtain tumor cells, irradiating the tumor cells to be viable but non-tumorigenic, and using these cells to prepare a vaccine for the patient capable of inhibiting recurrence of the primary tumor while simultaneously inhibiting metastases. The patent teaches the development of monoclonal antibodies which are reactive with surface antigens of tumor cells. As set forth at col. 4, lines 45 et seq., the patentees utilize autochthonous tumor cells in the development of monoclonal antibodies expressing active specific immunotherapy in human neoplasia.
U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic of human carcinomas and not dependent upon the epithelial tissue of origin.
U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies which induce apoptosis in Her2 expressing cells, hybridoma cell lines producing the antibodies, methods of treating cancer using the antibodies and pharmaceutical compositions including said antibodies.
U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for the production of monoclonal antibodies to mucin antigens purified from tumor and non-tumor tissue sources.
U.S. Pat. No. 5,869,268 is drawn to a method for generating a human lymphocyte producing an antibody specific to a desired antigen, a method for producing a monoclonal antibody, as well as monoclonal antibodies produced by the method. The patent is particularly drawn to the production of an anti-HD human monoclonal antibody useful for the diagnosis and treatment of cancers.
U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments, antibody conjugates and single-chain immunotoxins reactive with human carcinoma cells. The mechanism by which these antibodies function is two-fold, in that the molecules are reactive with cell membrane antigens present on the surface of human carcinomas, and further in that the antibodies have the ability to internalize within the carcinoma cells, subsequent to binding, making them especially useful for forming antibody-drug and antibody-toxin conjugates. In their unmodified form the antibodies also manifest cytotoxic properties at specific concentrations.
U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumor therapy and prophylaxis. However, this antibody is an antinuclear autoantibody from an aged mammal. In this case, the autoantibody is said to be one type of natural antibody found in the immune system. Because the autoantibody comes from "an aged mammal", there is no requirement that the autoantibody actually comes from the patient being treated. In addition the patent discloses natural and monoclonal antinuclear autoantibody from an aged mammal, and a hybridoma cell line producing a monoclonal antinuclear autoantibody.
U.S. Pat. No. 5,850,854 discloses a specific antibody, BR110 directed against GA733-1. This patent discloses in vitro function for BR110 as an immunotoxin conjugate. There was no in vitro function as a naked antibody disclosed for this antibody. There was also no in vivo function disclosed for this antibody.
U.S. Pat. No. 6,653,104 claims immunotoxin-conjugated antibodies, including but not limited to RS7, directed against a host of antigens, including but not limited to EGP-1. The immunotoxin is limited to those possessing ribonucleolytic activity. However, the examples disclose only a specific immunotoxin-conjugated antibody, LL2, directed against CD22. There was no in vitro or in vivo function for RS7 disclosed in this application.
U.S. Application No. 20040001825A1 discloses a specific antibody, RS7 directed against EGP-1. This application discloses in vitro function for RS7 as a radiolabeled conjugate. There was no in vitro function as a naked antibody disclosed for this antibody. This application also discloses in vivo function for RS7 resulting from radiolabled and unlabeled conjugate administered sequentially. However, this study was limited to one patient and it is unknown whether any of the observed function was due to the unlabeled antibody. There was no in vivo function for RS7 resulting from the administration of the naked antibody.

Cytotoxicity mediation of cells evidencing surface expression of TROP-2


A basket and mounting arrangement for use in a refrigerator/freezer storage compartment is disclosed. The basket is provided with a plurality of generally longitudinally extending support rails which are carried on each side of the basket and which are adapted to be slidably received in a pair of guide members secured to opposing sidewalls of the storage compartment. In one embodiment, the plurality of support rails comprise a first pair which are located on opposite sides of the basket in a substantially horizontal first plane and a second pair of support rails which are located on opposite sides of the basket and are vertically spaced a predetermined distance from the first pair of support rails in a substantially horizontal second plane. Either pair of support rails can be readily engaged with the pair of guide members to alter the vertical position of the basket. The basket is constructed in a manner such that the support rails are laterally spaced a predetermined distance from the sidewalls of the basket in order to prevent interference with the movement of the basket by goods stored therein. The guide members are integrally formed with upstanding abutment flanges which engage stop members carried by the support rails to limit sliding movement of the basket.

1. Field of the Invention
The present invention pertains to a basket and mounting arrangement for use in a refrigerator/freezer storage compartment. More specifically, the invention is directed to an improved basket arrangement for use in storing goods in a refrigerator/freezer storage compartment and a mounting assembly for slidably and vertically adjustably supporting the basket within the storage compartment in a manner which maximizes the storage capacity of the basket while assuring that any goods stored in the basket will not interfere with the selective movement of the basket.
2. Description of the Prior Art
Slidably mounted refrigerator/freezer storage compartment baskets are known in the art. Such known arrangements are generally mounted within the storage compartment upon a pair of guide rails such that the bottom of the basket simply rests atop the rails. Unfortunately, these known slidably basket mounting assemblies do not provide for readily and vertically adjusting the position of the baskets within the storage compartment.
It is further known in the art to slidably mount a basket to a pair of spaced guide rails secured within a storage compartment by utilizing a plurality of rollers which are attached to the basket and engage the guide rails. Although this type of arrangement provides for smooth operation of the basket into and out of the storage compartment, such a mounting arrangement requires rather precise tolerances in order to assure the smooth operation of the mounting assembly and also significantly adds to its cost. In addition, unless auxiliary rollers or guide rails are utilized, these known arrangements also do not provide for vertically adjusting the position of the basket within the storage compartment.
Finally, it is also known in the art to provide baskets with longitudinally extending flanges that project outwardly along a top edge of the basket's sides which can be used in combination with a pair of guide rails mounted in the storage compartment to enable the basket to be slidably mounted therein. In these arrangements, it is often found that goods stored in the basket interfere with the smooth operation of the slidable mounting assembly, especially when wire baskets are utilized.
Therefore, there exists a need in the art for a basket and mounting arrangement for use in the refrigerator/freezer storage compartment which will enable the basket to be readily slidable and vertically adjustable within the compartment in a cost-effective manner while assuring that any goods stored in the basket will not interfere with the movement of the basket.

Slidable and vertically adjustable refrigerator/freezer basket and mounting arrangement therefor


An animal food product useful as food supplement for pets such as dogs or cats, comprising a carrier and at least one food additive selected from vitamins, provitamins, minerals and trace elements, whereby the carrier comprises a cultured milk product.

The present invention relates to an animal food product useful as food supplement for pets such as dogs or cats. In particular, the present invention relates to a pet food supplement comprising a specific carrier and one or more food additives selected from vitamins, provitamins, minerals and trace elements. Moreover, the present invention relates to a process for the preparation of the food product and to specific uses of the food product.
Pets such as dogs and cats are most often fed with processed pet food products. The use of processed pet food products is problematic. In case mainly processed pet food products are fed it is likely that the diet of the pet will give rise to health problems. Such health problems may be due to the presence of ingredients having a negative effect on the digestion of the animal or due to the lack of certain vitamins, provitamins, amino acids, minerals or trace elements essential for restoring, preserving or improving the health of the animal. Health problems which may be associated with the consumption of processed pet food products are diarrhea, congestion, flatulence, lack of appetite, vomiting, loss of hair, unspecific itching of the skin, or skin diseases.
Therefore, it is desirable to supplement the diet of a pet in order to prevent or retract health problems or to improve the health and well-being of the pet. A food supplement is even more desirable in the light of the fact that pets such as cats and dogs are frequently kept inside the home of the respective pet owner. In the domestic environment, pets may become a burden in case of health problems due to the difficulties of keeping the domestic environment clean when the pets are ill. Specifically, in case of diarrhea, vomiting, and loss of hair the pet will likely cause damage in the domestic environment by polluting carpeting and upholstery. In case of flatulence and skin disease, the smell of the pet may become a burden in the domestic environment. Moreover, any health problems experienced by the pet will be likely to lead to undesireable behavioral changes of the pet.
Any pet food product including pet food supplements must meet in combination a number of different requirements to be commercially useful.
The food product must have a smell and flavor which render the food acceptable to the animal. In case smell or flavor are not acceptable for a specific animal, the animal will refuse to consume the food product. It is known to add flavoring agents as well as aroma and flavor enhancers to pet food products. However, a general acceptance among dogs or cats cannot be achieved with known substances. Usually, the acceptance of a common dry dog food product is in the order of 70%. In any case, it must be borne in mind that food products may not contain sugar as a flavoring agent in amounts which will lead to severe conditions such as blindness. Food additives such as certain vitamins to be administered to a pet may present an unacceptable taste to the pet. Therefore, it is often difficult to administer such food additives by simple admixing to the regular pet food since the unacceptable taste or smell cannot be efficiently masked by the regular pet food. In particular, the administration of food additives in high concentrations is problematic.
Animal food products must not have undesired effects on the health of the animal. In particular, the food product must not have adverse effects on the digestion of the animal such as causing congestion or diarrhea. In case such undesired effects on the digestion are observed, additional measures will be necessary in order to retract or avoid such undesired effects.
The food product must have an acceptable shelf life at room temperature since animal food products are sold in supermarkets in a designated pet food department which usually does not offer the possibility of cooling the merchandise. For the animal food products to be useful it is necessary that it may be stored for at least three months at room temperature without spoiling. Preferably, the shelf life is obtainable without the use of any preservative.
In case food additives are administered to a pet, the dosage of such food additives must be easy to control, in particular in cases where an overdosing with potentially harmful substances may lead to adverse effects on the health of the pet.
Accordingly, it is a problem for the present invention to provide an animal food product useful as a pet food supplement which
(i) enjoys high acceptance with pets,
(ii) has beneficial effect on the digestion of the animal,
(iii) has a shelf life without use of preservatives or cooling of several months, and which
(iv) provides the possibility for a simple and safe dosage of any food additive contained in the food supplement.
A further problem of the invention is to provide a process for the preparation of an animal food product useful as a pet food supplement which has the above properties (i) to (iv).
A still further problem of the invention is to provide the use of the animal food product of the invention in pet food and as a pet food supplement.
These problems are solved according to claim 1 with an animal food product useful as food supplement for pets such as dogs or cats, comprising a carrier and at least one food additive selected from vitamins, provitamins, minerals and trace elements, whereby the carrier comprises a cultured milk product.
The present invention provides a wet food product comprising a carrier and at least one specific food additive. It was found that surprisingly an animal food product containing food additives and having a very high acceptance with animals such as dogs and cats, may be prepared by using a specific wet carrier. The carrier is a cultured (fermented) milk product prepared from milk having a fat content of up to 2.5% by weight, preferably up to 2% by weight.
The present invention also provides a process for the preparation of an animal food product comprising the steps of adding at least one food additive selected from the group of vitamins and provitamins, minerals, and trace elements to milk having a fat content of up to 2.5% by weight, fermenting the mixture in the presence of a microorganism, and heating the mixture to an elevated temperature for a predetermined period of time to a predetermined temperature for essentially terminating the fermentation.
It is essential that the respective food additives are added. to the milk prior to the fermentation whereby the food additives may be effectively dissolved or dispersed in the milk and the shelf-life and acceptability of the product of the invention become superior. The specific reasons for the observed effects are presently not understood in detail. However, it is contemplated that the food additives will be at least partially incorporated into microorganisms or bonded to the microorganisms used for culturing during the fermentation process or to ingredients of the milk. Thereby, the taste and smell of the food additives is effectively masked and the desireable milk character in the taste and smell of the product is preserved. Moreover, it is contemplated that the incorporation or bonding of the food additives to the microorganisms or ingredients of the milk leads to a desireable slow release of the incorporated or bonded food additives in the digestive system of the animal.
The food product of the invention comprising a cultured milk product as a carrier has highly beneficial effects on the digestion of the animal whereby undesired side effects from the use of conventional processed animal food may be avoided, retracted and the digestion of the animals may even be improved.
The shelf-life of the animal food product is provided by heating the animal food product to a predetermined temperature for a predetermined time for essentially terminating the fermentation process and by storing the animal food product under essentially sterile conditions. The food product of the invention may be stored in packaged form without cooling or the use of preservatives, preferably for at least six month. Thus, the pet food product of the invention may be stored or offered in places which do not provide cooling for pet supplies, such as supermarkets or pet supply stores. Moreover, the food product of the invention does not have to be kept in a refrigerator by the pet owner. Thus, the animal food product may be kept separate from the food for human beings. Hence, undesirable confusion between food for humans and animals may be avoided.
The fermentation process used to prepare the animal food product of the invention provides a product having a viscous consistency which can easily be handled, wherein the food additives are well dispersed and any undesireable taste of a food supplement is effectively masked by a taste having a milk character preferred by pets. The use of the specific carrier of the invention allows to provide easy and predetermined dosage of the food additives based on the amount of the food product fed to the animal.
The shelf-life of the animal food product of the invention may be further extended by adding to the animal food product any preservatives conventionally used for preserving milk and dairy products.
The invention further provides the use of the animal food product of the invention in pet food and as a pet food supplement.
The animal food product of the invention comprises a carrier and at least one food additive.
The carrier is a cultured (fermented) milk product. The cultured milk product is a milk product prepared from partially skimmed milk, skim milk or reconstituted non-fat dry milk. Preferably, the milk is cow milk. It is essential that the fat content. in the milk used for the carrier of the animal food product of the invention is up to 2.5% by weight, preferably up to 2% by weight and more preferably about 1.5% by weight. If the amount of fat is above this range, the animal will be likely to suffer from diarrhea when ingesting the animal food product of the invention.
The milk product may be a product fermented with one or more bacterial cultures used in the art as starters for the preparation of cultured milk products. Specific examples of the starters are Lactobacillus bulgaricus, Streptococcus thermophilus, Streptococcus lactis, Streptococcus diacetilactis, Leuconostoc citrovorum, Lactobacillus acidophilus, Lactobacillus bifidus, Lactobacillus caucasieus.
In a preferred embodiment, the cultured milk product used as a carrier according to the present invention is a yogurt. The yogurt is preferably made by fermenting milk with Lactobacillus bulgaricus and Streptococcus thermophilus. The yogurt may be a set type yogurt which is filled immediately after the inoculation with bulk starter and incubated in the packages. The yogurt may also be a stirred type yogurt which is inoculated and incubated in a tank. After incubation the product is filled in packages. The heating for providing the shelf-life may be carried out before or after filling the product into the packages. In case the product is heated before filling, care must be taken during the filling operation in order to avoid contamination of the product whereby the shelf-life is deteriorated. Preferably, the product of the invention is a set type yogurt.
The consistency of the cultured milk product used as carrier according to the invention may range from a thin or creamy product to a gelatinous or firm pudding-like product depending on the preferences of the pet owner and the pet.
The animal food product according to the invention contains at least one food additive in an amount higher than the natural content or proportion of this food additive in the milk used for the preparation of the carrier. A food additive is a substance with nutritive and other dietary effects. Specifically, the food additive of the invention is selected from vitamins and provitamins, minerals and trace elements. In a preferred embodiment, the animal food product contains a mineral such as calcium and at least one vitamin or provitamin.
Any water soluble or fat soluble vitamins and provitamins are suitable for use in the animal food product of the invention. Examples for vitamins useful as food additives according to the present inventions are vitamin A, B1, B2, B6, B12, C, D, E, H (biotin), K, carotene, folic acid, inositol, niacin, and pantothenic acid. Preferred vitamins are vitamins A, D3, E, B6 and biotin. According to the most preferred embodiment of the invention these preferred vitamins are used in combination. Vitamin A is needed by pets for maintaining visual senses as well as healthy mucous membranes. Vitamin A deficiency results in skin diseases and in an increased susceptibility to infections. Vitamin A may be contained in the product of the invention in an amount of from 100 to 20,000, preferably 5,000 to 15,000, more preferably about 14,000 IU/kg of the fermented milk in the product.
Vitamin D3 stimulates the absorption from the bowel of calcium and phosphorous compounds, which are essential for the formation of the skeleton. Vitamin D3 may be contained in an amount of from 100 to 3,000, preferably 1,000 to 2,000, more preferably about 1,500 IU/kg of the fermented milk in the product.
Vitamins A and D must be controlled in order to avoid hypervitaminosis caused by the storage of these vitamins by the body of the animal.
Vitamin E is an anti-oxidant necessary for maintaining cell membranes and normal enzyme functionality. In case of vitamin E deficiency, cardiac and skeletal muscles degenerate, the physical development is disturbed and optomeninx degeneration is observed. Vitamin E is contained preferably in an amount of from 10 to 250 mg/kg, more preferably about 50 mg/kg of the fermented milk in the product.
Vitamin B6 (pyridoxal, pyridoxamine, pyridoxine) is essential for numerous reactions in the amino acid metabolism. Vitamin B6 deficiency results in a loss of weight and anemia of the animal. Vitamin B6 is contained preferably in an amount of from 2 to 20 mg/kg, more preferably about 12 mg/kg of the fermented milk in the product.
Biotin is essential for the carbohydrate, fat and protein metabolism. Biotin deficiency results in dermatitis as well as discoloration and loss of hair. Biotin is contained preferably in an amount of from 100 to 800 xcexcg/kg, more preferably about 460 xcexcg/kg of the fermented milk in the product.
Minerals and trace elements suitable for the animal food product of the invention are in particular calcium, magnesium, copper, zinc, and iron salts.
The animal food product of the present invention may further contain additives commonly known in the art in an amount which does not impair the purpose and effect provided by the invention. Examples of such additives are bulking agents, substances with a stabilizing effect, organoleptic substances, and processing aids.
Examples of bulking agents optionally contained in the animal food product of the invention are water, air, crystalline cellulose, or polydextrose.
Substances with a stabilizing effect may be added to increase the shelf life of the product of the invention by supplementing or reinforcing the effect of the physical methods used for increasing the shelf life according to the invention. Examples of substances with a stabilizing effect optionally contained in the animal food product of the invention are preservatives, antioxidants, synergists and sequestrants, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants. Examples for the emulsifiers and/or thickening agents are gelatine, cellulose ethers, starch, starch esters, starch ethers, modified starches, with modified starches being preferred.
Organoleptic substances may be used to stimulate the appetite of the pet by improving the product of the invention in view of appearance, taste, smell, and aroma. Such substances may be used to influence positively the senses of smell, taste, and vision before, during or after the consumption of the product by the animal. Examples of organoleptic substances optionally contained in the animal food product of the invention are coloring agents, color stabilizers, acidulants, substances with a salty taste, substances with a bitter taste, substances with an alkaline taste, flavor enhancers, spices and flavorings. A flavoring agent or an aroma or flavor enhancer may be added to the animal food product of the invention in particular when the animal food product is used in cat food.
Processing aids may be used to fulfill a certain technological purpose during treatment or processing. Examples of processing aids besides bacterial cultures which are optionally contained in the animal food product of the invention are enzymes and acidity regulators.
The process for the preparation of an animal food product according to the present invention comprises the steps of adding food additives selected from the group of vitamins and provitamins, minerals, and trace elements to milk having a fat content of up to 2.5% by weight, fermenting the mixture in the presence of a microorganism, and heating the mixture to an elevated temperature for a predetermined period of time to a predetermined temperature for essentially terminating the fermentation.
Now a preferred process for the preparation of the animal food product of the invention is described.
Food additives are added in the desired amounts to standardized milk having a content of fat of up to 2.5% by weight. The mixture is mixed whereby the food additives are thoroughly dissolved or dispersed in the milk. Optionally, standardization of the milk may be carried out after the food additives were added whereby the solids content is adjusted in order to improve the structure of the finished product. Any other optional component such as emulsifiers and thickening agents may also be added at that stage.
The water content of the mixture is adjusted to provide a cultured milk product having a water content of at least 70% by weight, preferably at least 80% by weight.
Preferably, the mixture is then subjected to a thermal treatment and/or homogenization.
The thermal treatment may comprise thermization, pasteurization, sterilization or ultrahigh temperature heating for the purpose of the destruction of microorganisms and the provision of physicochemical changes such as inactivation of enzymes, denaturation of whey proteins, activation of sulfhydryl groups, and liberation of hydrogen sulfide, and the Maillard reaction between protein and lactose. However, it must be kept in mind that thermal treatment of the milk may destroy vitamins. Therefore, excess amounts of thermally labile vitamins may be used to account for any loss during thermal treatment of the milk. Moreover, the process conditions may be adjusted to minimize any loss of vitamins.
Homogenization may be used for providing a uniform fat distribution, a whiter color, a fuller taste, and a softer coagulum on clotting and consequently a better digestability of the animal food product of the invention. When homogenization is applied, the average particle size of the fat globules is reduced to below 2 xcexcm, preferably below 1 xcexcm.
Subsequently, starter cultures are added to the mixture. The starter cultures may be liquid or freeze-dried starting cultures according to common practice in the art.
In a first preferred embodiment, the mixture is then filled into packages and sealed under essentially sterile conditions. The package may be any package capable of being heated to a temperature for a predetermined time for essentially terminating the cultivation of the mixture. Preferably a plastic package such as a plastic cup having a functional coating on the inner wall is employed. Such functional coating as known in the art should not be detrimental to the mixture and the food product in a temperature range of from xe2x88x9250xc2x0 C. to 100xc2x0 C. The plastic cup may be sealed with a metal or plastic foil according to methods generally known in the art.
The packaged mixture is then incubated preferably at a temperature of about 40 to 45xc2x0 C. for 1 day for fermentation of the mixture whereby the pH of the mixture is lowered in a range of pH 3.9 to 4.5.
After culturing, the packaged mixture is heated to elevated temperatures for essentially terminating the fermentation. Preferably, the packaged mixture is heated to a temperature of from 60 to 75xc2x0 C., preferably to about 70xc2x0 C. for a period of 1 to 10 minutes, preferably for about 4 minutes. The thermal treatment increases the shelf life of the final product to more than three month. Finally, the packaged product is cooled in order to provide an animal food product of the invention.
In a second preferred embodiment, the mixture is then kept in a fermenter. The mixture is incubated in the fermenter preferably at a temperature of about 40 to 45xc2x0 C. for 1 day for fermentation of the mixture whereby the pH of the mixture is lowered in a range of from pH 3.9 to 4.5.
Subsequently, the fermented mixture is filled into packages under essentially sterile conditions. The package may be the same as in the first preferred embodiment.
After filling the fermented mixture into packages, the packaged mixture is heated to elevated temperatures for essentially terminating the fermentation. Preferably, the packaged mixture is heated to a temperature of from 60 to 75xc2x0 C., preferably to about 70xc2x0 C. for a period of 1 to 10 minutes, preferably for about 4 minutes. The thermal treatment increases the shelf life of the final product to more than three month, typically to at least six month. Finally, the packaged product is cooled in order to provide an animal food product of the invention.
In the process for preparing an animal food product of the invention it is essential that the food additives selected from vitamins, provitamins, minerals and trace elements are added to the milk prior to fermentation. In case the food additives are added after fermentation, the shelf life of the product will deteriorate. Moreover, the presence of the food additives during fermentation will lead to the incorporation of the food additives in the microorganisms used for fermentation whereby any unacceptable flavor or smell of the food additives may be effectively hidden. Further, an excellent dissolution or dispersion of the food additives within the cultured milk product may be obtained.
The animal food product may contain any food additives in any desired amount as long as the dosage is high enough to provide a desired effect and provided that the dosage is low enough to avoid an impairment of the purpose or effect of the invention.
Usually, the food additives are dosed and administered to an animal based on the amount of the animal food product depending on the type, sex, weight, health, and age of the specific animal and the frequency of administration.
The food additives in the animal food product of the invention may be administered in admixture with the regular animal food or as a separate feed. The animal food product may have any temperature when administered to the animal. Usually room temperature is preferred. However, the administration of the animal food product of the invention as a frozen yogurt type product is contemplated.
Vitamin A may be administered to a pet by using the animal food product of the invention in an amount of from 100 to 20,000, preferably 1,000 to 15,000, more preferably about 1,700 to about 12,000, most preferably about 3400 IU per week.
Vitamin D3 may be administered to a pet by using the animal food product of the invention in an amount of from 100 to 3,000, preferably about 160 to about 1,100, more preferably about 320 IU per week.
Vitamin E may be administered to a pet by using the animal food product of the invention in an amount of from 10 to 250, preferably 12 to 100, more preferably about 14 to about 96, most preferably about 28 mg per week.
Vitamin B6 may be administered to a pet by using the animal food product of the invention in an amount of from 0.5 to 20, preferably 1 to 8, more preferably about 2.3 mg per week.
Biotin may be administered to a pet by using the animal food product of the invention in an amount of from 100 to 800, preferably 58 to 400, more preferably about 115 xcexcg per week.
Calcium may be administered to a pet by using the animal food product of the invention in an amount of from 100 to 1,000, preferably about 140 to 950, more preferably about 250 mg per week.
The above ranges refer to an adult dog having a body weight of about 8 to 10 kg. The dosage may be adapted in case of a deviation of the body weight, maturation or type of a specific animal. In particular, problems associated with hypervitaminosis should be avoided by the adjustment of the dosage.
In the most preferred embodiment, an animal food product having a predetermined composition may be packaged in cups containing the product in an amount of 125 g per cup. Such cups may be fed in an amount of from 1 to 7 cups per animal per week. The composition of the product should be adjusted so that the weekly dosage administered to the animal is within the preferred ranges given above. Accordingly, the dosage and administration of the animal food product of the invention is easy and reliable in particular for elderly pet owners.
Now the invention will be described with reference to specific examples.


A finite difference wavefield modeling framework decouples the tasks of physical modeling and hardware-software optimization through the use of a platform-agnostic intermediate representation in the form of a dataflow graph. In at least some embodiments a wavefield simulation method includes specifying a kernel of operations to be applied at each point in a space and representing the kernel as a platform-agnostic dataflow graph. For each of multiple implementation platforms, the method further includes: deriving from the platform-agnostic graph a customized graph for the implementation platform; translating the customized graph into configuration information for the implementation platform; supplying the configuration information to the implementation platform to obtain a wavefield simulator; and employing the wavefield simulator to repeatedly apply said kernel of operations to each point in the space with specified input or output signals.

Scientists and engineers often employ geophysical surveys for exploration, archeological studies, and engineering projects. Geophysical surveys can provide information about underground structures, including formation boundaries, rock types, and the presence or absence of fluid reservoirs. Such information greatly aids searches for water, geothermal reservoirs, and mineral deposits such as hydrocarbons and ores. Oil companies in particular often invest in extensive seismic and electromagnetic surveys to select sites for exploratory oil wells.
Geophysical surveys can be performed on land or in water. As indicated in FIG. 1, an energy source 102 near the region of interest 104 generates waves 106 that propagate into the region of interest and reflect from internal features such as bed boundaries. Eventually, the reflected waves 108 reach an array of receivers 110 on or near the surface 112 or in a borehole. A recording system 114 captures the received signals for storage and processing. The process is repeated with many different source positions and optionally with different receiver positions. Although various methods exist for converting the received wave signals into an image of the subsurface structure, the most popular such techniques employ finite difference wave field modeling, a process that propagates waves forward or backward in time using discrete time steps and fast approximations of wave function derivatives. With small enough time steps and accurate enough approximations, the finite difference wave field modeling process provides an efficient, high fidelity solution. Nevertheless, the finite difference modeling process imposes a substantial computational demand.
Many efforts have been made to satisfy the computation demands of the finite difference wave field modeling process. Many combined hardware-software solutions have been implemented in the attempt to maximize runtime efficiency. In addition to exploiting different parallelization strategies offered by different hardware systems, the software is often customized to incorporate different earth models, different wave function approximations, and different propagation strategies. These factors lead to a severe fragmentation of the code base, making it difficult to maintain and re-use applications. In many cases, it may be difficult to even compare the results of different applications due to slightly different underlying algorithms or hardware implementations.
It should be understood, however, that the specific embodiments given in the drawings and detailed description below do not limit the disclosure. On the contrary, they provide the foundation for one of ordinary skill to discern the alternative forms, equivalents, and other modifications that are encompassed in the scope of the appended claims.

Unified application framework for finite-difference modeling


The present invention relates to a method for verifying concrete flexural strength by establishing a predictive correlation between concrete flexural strength and a second strength parameter, such as direct tension or indirect tension, measuring the value of that second strength parameter for a batch of concrete, and estimating the flexural strength of the batch by way of the second strength value and the correlation.

Concrete pavement design relies upon the flexural strength of the concrete as a primary design input. Concrete flexural strength is difficult to measure precisely due to the inherent problems associated with casting and handling the relatively large beam specimens required by the test procedure (e.g. according to ASTM C78). Because of this difficulty, many state highway agencies rely upon cylinder specimens tested in compression (e.g. according to ASTM C39) to control concrete quality in the field. However, the failure mechanisms and causes of failure can be considerably different between compressive-strength test methods and flexural-strength testing.

Method for verifying concrete flexural strength


A method for fabricating a semiconductor device is provided. The method includes: providing a first wafer having a first active surface and a first rear surface opposite to the first active surface, the first wafer comprising a first circuit formed therein; providing a second wafer having a second active surface and a second rear surface opposite to the second active surface, the second wafer comprising a second circuit formed therein; bonding the first active surface of the first wafer with the second active surface of the second wafer so as to electrically connecting the first circuit and the second circuit; thinning the second wafer from the second rear surface; and forming at least a conductive through via in the second wafer, wherein the conductive through via is electrically connected to the first circuit through the second circuit.

In the field of semiconductor packaging, a through silicon via (TSV) is mainly used for allowing wires on the top surface and the bottom surface to be electrically connected to each other. Distinguished from the conventional semiconductor packaging, through the disposition of the TSV, a plurality of chips within a semiconductor packaging may provide an enhancement of the packaging density of the semiconductor packaging structure, a reduction of the size of the semiconductor packaging structure, an increment of the speed of the device, a reduction of signal delay and power consumption. Accordingly, the development of the TSV technology is advantageous for electronic product miniaturization due to the semiconductor packaging industrial attention to the TSV.
In present, the manufacturing process of the TSV is normally performed after the manufacture of a semiconductor device layer in the semiconductor manufacturing process. In general, a through hole is required to be formed on a wafer during the manufacture of the TSV. For example, the required through hole for forming the TSV may be achieved by an etching process on the back side of the wafer until the formed through hole exposes a complementary metal-oxide semiconductor. Alternatively, the through hole may be formed by an etching process on the front side of the wafer until the through hole extends to the back side of the wafer. The technique of etching the wafer from the front side may possibly damage a metal-oxide semiconductor while the technique of etching the wafer from the back side may prevent an adverse impact on the complementary metal-oxide semiconductor. Therefore, the technique of etching the wafer from the back side to form the through hole grows and becomes mainstream. Nonetheless, due to a higher aspect ratio of the through hole penetrating the wafer, when to stop the etching procedure is difficult to be determined. Hence, the researchers may encounter difficulties in controlling the etching depth of the through hole so that the wafer may not be completely etched or may be over-etched at which the through hole is located. When the through hole is not etched completely, the wires in the complementary metal-oxide semiconductor layer may not be exposed by the through hole and may not be connected to a conductor in the through hole. Thus, an open circuit occurs between the wires and the conductor in the through hole. When the over-etching occurs, notches may be formed at the interface between the substrate and the complementary metal-oxide semiconductor device layer in the wafer, the notches may lead to difficulties in the deposition of an insulting liner layer. For example, the deposition thickness of the insulting liner layer on the notches may be insufficient or the insulting liner layer may be non-continuous on the notches, and therefore the notches may result in electrical leakages. The reliability and the manufacturing yield of the semiconductor structure may be adversely affected.
In view of the foregoing, to properly control the process recipe of a conductive through via to prevent the aforementioned notches during the manufacturing process becomes an important issue.

Semiconductor structure and manufacturing method thereof

Industrial Technology Research Institute


A new and distinct cultivar of Osteospermum plant named ‘Balostlueye’, characterized by its medium yellow and dark purple colored inflorescences, medium green-colored foliage, and moderately vigorous, upright-mounded growth habit, is disclosed.

The present invention relates to a new and distinct cultivar of Osteospermum plant botanically known as Osteospermum ecklonis and hereinafter referred to by the cultivar name 'Balostlueye'.
The new cultivar originated in a controlled breeding program in Santa Paula, Calif. during July 2011. The objective of the breeding program was the development of Osteospermum cultivars that are freely flowering with unique flower coloration and a freely branching, upright-mounded growth habit.
The new Osteospermum cultivar is the result of cross-pollination. The female (seed) parent of the new cultivar is the proprietary Osteospermum ecklonis breeding selection coded 49249-5, not patented, characterized by its dark yellow and lavender colored inflorescences, medium green-colored foliage, and moderately vigorous, compact-mounded growth habit. The male (pollen) parent of the new cultivar is the proprietary Osteospermum ecklonis breeding selection coded 49305-4, not patented, characterized by its light yellow-colored inflorescences that age to a frosted purple, medium green-colored foliage, and moderately vigorous, mounded growth habit. The new cultivar was discovered and selected as a single flowering plant within the progeny of the above stated cross-pollination during April 2012 in a controlled environment in Santa Paula, Calif.
Asexual reproduction of the new cultivar by terminal stem cuttings since April 2012 in Santa Paula, Calif., Arroyo Grande Calif., and West Chicago, Ill. has demonstrated that the new cultivar reproduces true to type with all of the characteristics, as herein described, firmly fixed and retained through successive generations of such asexual propagation.


A microprocessor-controlled remote resistance measurement system is disclosed wherein the connection leads to the three- or four-wire resistance temperature devices (RTDs) are multiplexed via a four-channel analog multiplexer at the input of the unit. A separate two-channel multiplexer is also used to multiplex a fifth input for measurement of a reference resistor. The output of the multiplexer is coupled to a voltage-to-frequency converter, wherein the frequency output is utilized as an input to the microprocessor-based controller. The microcontroller can check for broken wires by addressing the multiplexers to individually isolate any of the connecting wires to the remote RTD sensors. The output of the multiplexer is monitored in a Test Mode by connecting a known impedance to the multiplexer output to determine if any RTD connections are defective. If one of the voltage sensing wires is faulty, the known impedance will cause an erroneous frequency reading into the microcontroller, which will then provide an indication on the display for determining exactly which RTD wire is broken. Only two address lines are used to control the five multiplexer channels through the use of a function selector circuit and a two-stage measurement cycle.

Two-wire transmitters are commonly used to monitor various conditions at remote locations. For example, to measure the liquid level in a tank at a remote processing plant from its central control room, a two-wire transmitter at the remote location is typically connected in series with a power supply and a load at a central location through two transmission wires. As the condition being monitored by the transmitter varies, the effective series resistance across the transmitter also varies so as to produce a corresponding change in the current drawn by the transmitter. An industry standard has developed in a large number of applications, wherein the current through the two-wire transmitter loop varies from 4-20 milliamperes (mA), wherein 4 mA is the minimum amount of current required to power the remote transmitter.
Volume, pressure, liquid level, and temperature are just some of the conditions which are typically monitored using two-wire transmitters. Temperature, however, is one of the conditions which often must be measured with precision. It is well known to utilize a resistance temperature device (RTD) for this purpose. The RTD is typically immersed in the medium, the temperature of which is to be measured, such that the resistance of the RTD will vary with the temperature changes of the medium. Utilizing either a table of resistance-temperature values or a polynomial equation to represent the relationship between the RTD's resistance and temperature, the actual temperature is then calculated from the measured resistance value of the RTD.
If the RTD is connected to the two-wire transmitter via two wire leads, then the RTD resistance measurement would necessarily include the resistance of the wire leads. For more accurate temperature measurements, a four-wire RTD system is often employed, i.e., two wires from each terminal of the RTD are connected to the two-wire transmitter. Two of the wires are used to pass current through the RTD, and the other two wires are used to sense the voltage developed across the RTD during the measurement. In this manner, the RTD's resistance is measured without passing current through the same wires that sense the voltages, i.e., without including the voltage drop of the lead wires. In still another version of an RTD system, a three-wire RTD is used, wherein such lead-length compensation is performed by measuring the voltage difference between only one voltage sensing lead and the current return lead. Numerous other RTD configurations are also possible, a few of which will be described below.
A problem often occurs whenever one of the wires to the RTD breaks or has an intermittent connection. Although a broken wire in the RTD's current path wires would immediately be apparent at the two-wire transmitter as an over-ranging, i.e., infinite, resistance measurement, a break in the voltage sensing wires may only slightly affect the resistance measurement by the amount of lead-length compensation being performed. In other words, depending upon the condition sensor configuration and the particular lead wire that is broken, a remote measurement system may appear to be functional yet be providing inaccurate readings for quite some time before the broken wire is discovered.
A need, therefore, exists for an improved remote measurement system which addresses the problem of detecting a broken wire in a three- or four-wire RTD temperature measuring unit.

Computerized remote resistance measurement system with fault detection


An induction heating coil used in a floating zone melting method, characterized in that the path of a high-frequency current is controllable in the body surface of the coil and by the use of the coil a more uniform resistivity profile across a diameter is achieved as compared with those from the prior art. In the coil a second metal or alloy different in electric conductivity from a first metal or alloy of the coil is disposed in a predetermined place(s) to control the path of a high-frequency current, wherein the second metal or alloy has, for example, higher electric conductivity than the first metal or alloy and a long narrow and thin strip made of the second metal or alloy is disposed in such a manner that it meanders on the body surface of the coil between the outer and inner peripheries periodically.

1. Field of the Invention
The present invention relates to an induction heating coil used as the heating device of a semiconductor single crystal grower and more particularly to an induction heating coil used for a crystal grower in which a dopant substance is uniformly dispersed in a grown crystal during the growth by FZ method (an abbreviation of the so-called floating zone melting method).
2. Description of the Prior Art
An apparatus used for growing a semiconductor single crystal by FZ method comprises an upper shaft for holding a raw material rod, a lower shaft for holding a seed crystal made of a single crystal semiconductor having a smaller diameter and a heating device arranged such that it causes the raw material rod to fuse by heating.
In growing a single crystal with the apparatus above-mentioned, an end of the raw material rod is fused to be connected to the free tip of the seed crystal to produce a molten zone and in succession a narrow single crystal rod portion is grown dislocation free on the seed crystal and then the raw material rod starts moving downward together with the seed crystal relative to the heating device to drive the molten zone toward the other end of its own along the rod, while being rotated about its axis and thereby almost the whole length is crystallized in the shape of a rod.
An apparatus for growing a crystal rod requires that a raw material is fully fused without any core portion left not fused within a narrow molten zone therein during zone melting. Therefore, as a heating device, which meets the requirement, for example, an induction heating coil having a construction of a single turn and being flat is well known in the prior art.
Such well known induction heating coils include, for example, an induction heating coil 1 as shown in FIG. 2, wherein a hollow conductor 4 is bent in the shape of a ring and both of the ends are located face to face with a gap 3 interposed therebetween in the closest relation that does not cause undesirable sparks with each other. The induction heating coil is electrically connected to a pair of power source terminals 5 at the two respective points on the outer vertical surface in the vicinity of both the coil ends and the section in parallel with the radial direction is in the shape of a wedge (the Japanese second publication No.51-24964, hereinafter referred to as the first prior art).
Such a construction of the coil of the first prior art may give rise to a current path almost a perfect geographic symmetry to the coil center in the surface of the coil. When a high-frequency current is supplied through the power source terminals 5 to the coil 1, an electromagnetic field is formed on and around the coil 1 and the field is almost uniform in strength in the area surrounded by the inner periphery 2, within which a raw material rod is vertically disposed therethrough and thereby the rod is partly heated to form a molten zone therein.
However, an induction coil of the first prior art has the end gap 3 being formed in parallel with a plane perpendicular to a tangent at the ends of the peripheries of the coil and thereby uniformity is caused in the vicinity of the gap 3. On the other hand, the high-frequency current supplied from the power source terminals 5 flows on and along the current path 7 depicted in FIG. 2 due to its nature, which takes a shortest path in principle. As a result, an induction coil 1 of this kind comes to give rise to regions 8 of a lower-density current in the surface thereof and thereby again causes another ununiformities thereon and therearound. In the mean time, the regions 8 are only in part shown in FIG. 2 for clarity.
Such an uneven magnetic field brings about an uneven heating capacity distribution over the coil and thereby a growing crystal is adversely affected in quality. For instance, when a raw material rod is rotated and transferred relative to the induction heating coil 1 in such a uneven magnetic field, a pair of a higher and the following lower layer in a dopant concentration is periodically produced in the growing portion within one revolution of the rod due to differentials of local heating capacity caused by the uniformity in the uneven magnetic field surrounding the coil. In one revolution of the rod, a dopant concentration of the growing front of the crystal becomes lower in a portion at a higher temperature and conversely the dopant concentration becomes higher in a portion at a lower temperature, which is called "periodical resistivity variation". Semiconductor wafers produced by slicing a single crystal rod having such a repetition in bulk of the combination of higher and lower concentrations as described above bring about fluctuations in characteristics of a semiconductor device fabricated starting therefrom due to microfluctuation in resistivity observed in the regions of periodical resistivity variations.
A contrivance to compensate the faults observed in the first prior art has been made, which is constructed in such a manner that as shown in FIG. 3, a single-turn flat coil 1 has two slits 6 extending from the inner periphery 2 towards the outer periphery 4 and further to the contrary another three slits 6 extending from the outer periphery in the reverse way (the Japanese first publication No.52-30705, hereinafter referred to as the second prior art). In this second prior art, the induction heating coil 1 has a plurality of slits 6 with all the widths thereof being same in dimension as that of the gap 3 at the ends of the coil 1. The slits 6 are arranged at equal distance intervals in the circumferential direction. The configuration of said slits has a geometrical periodicity about the center of the coil. Under such conditions a high-frequency current is controlled to flow in the surface of the coil 1 so as to keep an axial symmetry to the central axis of the coil.
Even an induction heating coil in the second prior art, however, is not able to avoid occurrence of lower current density regions 8 in the surface of the coil, as shown in FIG. 3. This phenomenon originates in the nature of a high-frequency current flowing a shortest path and in the structural restriction of the induction heating coil itself as well.
In particular, the induction heating coil 1 has to have such a configuration for cooling the coil that a coolant may flow inside of the coil having a hollow structure. In line with the requirement, narrow regions 9 should be provided at least both on the outer periphery side and on the inner periphery side of the coil, where the widths of the narrow regions 9 should be large enough to allow the coolant water to flow through. Consequently, the high-frequency current does not take a path along and in close vicinity to the inner periphery and along both the sides of slits and thereby the lower-density current regions 8 are created in the surface of the coil. With this configuration of the coil 1, the current path 7 is not able to be selected nearer enough toward the inner periphery and thereby a stirring power in the core portion of the molten zone becomes week, so that resistivities in the core portion of a semiconductor crystal rod thus produced are the lower corresponding to the decrease in the stirring power.

Induction heating coil suitable for floating zone processing


The present invention is a package for selectively heating or cooling a food product of the package utilizing a temperature-changing pack disposed within an inner container. The temperature-changing pack is formed in a manner which prevents the inadvertent or premature mixing of the chemical reagents to initiate the heating or cooling reaction. Further, the temperature-changing pack is positioned within the inner container at a location where the food product comes into contact with the pack on multiple sides to maximize the heat transfer between pack and the food product. The inner container can be positioned within a reusable outer sleeve that insulates the inner container to further maximize the heat transfer between the pack and the food product.

Currently, a large number of food products or items are packaged in a manner that enables the food products to be easily transported and/or consumed. While the majority of these types of packaging allow for the food product to be easily held within the container until it is desired to consume the food product, some of these containers do not provide the ability to heat or cool the food product as desired prior to consuming it.
Thus, in order to enable the container to heat the product or cool the food product as desired prior to consumption, a number of different heating and cooling mechanisms have been developed for use with containers of this type. One of the more economical ways used to heat or cool a food product within the container holding the food product is a chemical heating or cooling pack disposed in the container in direct contact with the food product to be heated or cooled. The chemical pack includes a number of chemical reagents contained within separated compartments of the pack that can be ruptured to allow the reagents to mix. When the reagents in the chemical pack are mixed, the resulting reaction functions to supply or remove heat energy, thereby heating or cooling the food product held within the container in contact with the chemical pack.
However, when chemical packs of this type are utilized, significant problems with the heating or cooling of the food product in the container are still encountered. For example, the chemical pack positioned within the container is often not located in an optimal location for effectively heating or cooling the entire food product. In most containers of this type, the chemical pack is located on one side of the container and/or is secured to the container completely around the periphery of the pack. In these configurations, the food product to be warmed or cooled does not contact the chemical pack around the entire pack, resulting in a significant amount of heating or cooling energy generated by the pack that does not come into contact with the food product.
Further, in order to enable an individual to place a food product into, or remove the product from the container, an opening is formed in the container. However, in most containers, the opening is large enough for the individual to reach into the interior of the container and come into direct contact with the chemical pack, potentially injuring the individual. Also, because the chemicals used as the reactants in the chemical pack could be harmful if ingested by an individual, should one or more of the components of the pack rupture, the reagents can mix with the food product without any indication that this has occurred. Further, depending upon the particular reagents utilized and the structure of the chemical pack in which the reagents are contained, if the reactants leak in small amounts into contact with one another prior to activation of the pack, a runaway reaction may occur in which the desired temperature for the reaction is greatly exceeded, such that the reaction can damage or completely burn through the container holding the food product.
As a result, it is desirable to develop a self-heating or self-cooling food product package or pouch that avoids these deficiencies present in prior art self-heating and self-cooling containers.


Some embodiments provide a method for a first managed forwarding element (MFE). The method receives a data message that includes a logical context tag that identifies a logical port of a particular logical forwarding element. Based on the logical context tag, the method adds a local tag to the data message. The local tag is associated with the particular logical forwarding element, which is one of several logical forwarding elements to which one or more containers operating on a container virtual machine (VM) belong. The container VM connects to the first MFE. The method delivers the data message to the container VM without any logical context. A second MFE operating on the container VM uses the local tag to forward the data message to a correct container of several containers operating on the container VM.

More and more applications are deployed into virtual machines (VMs), many of which consume network and security services (e.g., firewall, access control lists (ACLs), quality of service (Qos), etc.). In virtualized networks, virtualized systems may further virtualize other systems, increasing complexity and depth in a system creating a layer of virtual interfaces behind virtual interfaces. For example, Linux containers running on a VM may create several interfaces share a single interface of the VM (also referred to as a container VM). The container interfaces that share a single interface of the container VM may make it difficult to provide a ubiquitous network virtualization platform that provides network and security services for non-container VMs as well as for containers that are executing within a container VM in the system.


A cap is disposed internally of a spray nozzle and effects active atomization of pressurized liquid flowing through the nozzle by mixing with compressed air and creating turbulence in the liquid during the flow. The nozzle includes an internal cap which cap which imparts significant turbulence on the flow through the nozzle serving to help atomize the liquid using a reduced amount of air energy. Different caps may be used to create various selected spray patterns.

The invention relates to a spray nozzle and, more particularly, to a nozzle for directing a pressurized spray of atomized liquid into the atmosphere in the form of extremely small particles.
Systems for atomizing liquid with a pressurized gas such as air are known. In certain ones of such systems, the liquid is broken up both mechanically and by the pressurized air in an atomizing chamber located upstream of the spray nozzle. The atomized liquid then is ejected from the nozzle through one or more discharge openings in the nozzle.
An often-sought goal in atomizing and spraying apparatus is to achieve high efficiency. High efficiency in the context of the present invention refers to using as little air energy as possible to break liquid of a given volume into particles having a large total surface area. Larger surface areas are, of course, created by breaking the liquid into very fine particles.
A further goal is to provide nozzles having the capability of discharging the liquid in different spray patterns. By way of example, some applications require a narrow angle round spray, other applications may require a wide angle round spray (i.e., a full cone spray) and still other applications may require a flat spray.
In prior atomizing/spraying apparatus, the desired spray pattern is usually generated by forcing the atomized liquid through properly shaped discharge orifice means in the nozzle. A narrow angle round spray, for example, may be created by providing the nozzle with a single round orifice. A wide angle round spray may be generated by a nozzle having a plurality of angularly spaced diverging orifices. An elongated slot or an elliptically shaped orifice in the discharge nozzle produces a substantially flat spray pattern.
Nozzles having discharge orifices of the above type are essentially passive with respect to effecting further atomization of the liquid as the liquid is discharged from the nozzle. Certain nozzles do produce some further atomization during flow of the liquid through the nozzle but, for the most part, the atomization effected by the nozzle has limited impact on the overall efficiency of the atomizing and spraying apparatus.

Enhanced efficiency atomizing and spray nozzle


An electrical connector (12) has a body (20) with a shorting clip (26) mounted at a distal end (24) of the body (20). The clip (26) axially extends into a cavity (22) of the body and normally shorts two electrical male terminals (25) housed within the body (20).

Female electrical connectors commonly have a shorting clip in the form of a U-shaped body having two depending legs to engage the sides of the respective female terminals. When the male connector is plugged in, the male connector has an abutting surface that engages a ramp on the U-shaped body to deflect it away from the female terminals.
The known designs for shorting clips may be very sensitive to the overall tolerance stack of the connection system. Some designs, particularly ones with lift ramps, are also sensitive to the amount of axial movement of the electrical plugs. Some lift ramps do not always guarantee sufficient deflection to assure disengagement from the female electrical terminals.
Some clips may be overstressed when deflected to introduce permanent yielding that may result in, when released from the deflective force, loss of resilient motion back toward the electrical terminals with a resultant lack of adequate shorting contact force with the electrical terminals.
Shorting clip designs have required mounting of the clip axially within the electrical terminal body which requires specialized assembly steps often with specialized assembly tooling.


In an automotive hatch back vehicle, foldable bonnets are coupled between an underside of the hatch lid and the run-off channels on both the driver and passenger side of the vehicle, pleated to fold outwardly as the hatch lid is raised to divert water accumulations on the lid into the channels, and to fold away inwardly into the vehicle as the lid is lowered.

1. Field of the Invention
This invention relates to automotive hatch-back vehicles, in general, and to the drainage systems employed with their hatch lids, in particular.
2. Description of the Related Art
Automotive hatch-back vehicles are often designed with channels and weep holes to drain rainwater from their hatch lids. Experience has shown that some designs—such as those of the Pontiac Trans Am, the Chevrolet Camaro, the Ford Mustang and the Ford Escort—do not work very well; in them, when the hatch lid is raised, the rainwater gushes from the run-off channels into the interior of the vehicle, wetting the rear seat, the rear seat speaker installations, the carpeting in the hatch area and the rear seat belt retractors (which, in freezing temperatures, then become "locked" and inoperative). Experience has also shown that such channels exhibit a tendency to accumulate rainwater, so that even when the vehicle has dried, raising the hatch lid results in the accumulations dripping into the hatch area once more. This problem is particularly exasperating after the vehicle has been washed clean and then dried off—in that then raising the hatch lid cascades the car-wash water in, to damage the interior, to rust various panelings, and to ruin the owner's overall investment in the vehicle in general.