text:
The present inventors used antibody engineering techniques to prepare functional antibodies that correspond to individual mutations in causative genes of diseases, and discovered that such antibodies enable the treatment of the diseases. Specifically, the inventors succeeded in preparing ligands, particularly minibodies, which have agonistic activity to receptors that have almost completely lost responsiveness to their natural ligands because of gene mutations (for example, a thrombopoietin (TPO) receptor whose reactivity to TPO has been markedly impaired), and which can transduce signals by interacting with these mutant receptors at levels comparable to normal.
1. A minibody that acts as an agonist of a mutant human thrombopoietin receptor, the minibody comprising two or more antibody heavy chain variable regions (VH) and two or more antibody light chain variable regions (VL), each variable region being linked to at least one other variable region via a linker, wherein the minibody is a stronger agonist of the mutant receptor than is thrombopoietin, and wherein the mutant receptor includes at least one of the mutations R102P, R257C, and P275T in SEQ ID NO:2.
2. The minibody of
3. The minibody of
4. The minibody of
5. The minibody of
6. The minibody of
7. A pharmaceutical composition for treatment of a disease caused by a mutant human thrombopoietin receptor the composition comprising:
(a) a minibody that comprises two or more antibody heavy chain variable regions (VH) and two or more antibody light chain variable regions (VL), each variable region being linked to at least one other variable region via a linker, wherein the minibody is a stronger agonist of the mutant receptor than is thrombopoietin, and wherein the mutant receptor includes at least one of the mutations R102P, R257C, and P275T in SEQ ID NO:2; and
(b) a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of
9. The pharmaceutical composition of
10. The pharmaceutical composition of
11. The pharmaceutical composition of
12. The pharmaceutical composition of
13. An in vitro method comprising contacting the minibody of
14. The method of
15. The method of
16. The method of
17. The method of
18. The method of
19. The method of
20. The minibody of
21. The minibody of
22. The minibody of
23. The minibody of
24. The minibody of
25. The minibody of
26. The pharmaceutical composition of
This application is the National Stage of International Application No. PCT/JP2004/003334, filed Mar. 12, 2004, which claims the benefit of Japanese Patent Application Ser. No. 2003-067832, filed on Mar. 13, 2003. The contents of both applications are hereby incorporated by reference in their entireties.
The present invention relates to ligands having agonistic activity to mutant receptors, and pharmaceutical compositions comprising the ligands as active ingredients.
In recent years, the causative genes of various diseases have been identified in quick succession, and a variety of therapeutic methods for such diseases have been studied and established. Of these methods, the most intensively studied are mostly therapeutic methods for complementing enzyme gene deficiencies. It has been reported that enzyme replacement therapy using "Cerezyme" (Genzyme) is effective for patients with Gaucher's disease, in which β-glucocerebrosidase is deficient, and that enzyme replacement therapy using "Aldurazyme" (Genzyme) is effective for patients with mucopolysaccharidosis, in which α-L-iduronidase is deficient. Previously attempted gene therapies include introducing the adenosine deaminase (ADA) gene to patients with ADA deficiency, and introducing the coagulation factor IX gene to patients with hemophilia B. In addition to enzyme deficiencies, a large number of genetic diseases are known, such as genetic diseases of cytokines and their receptors. Some patients with type II diabetes mellitus, which accounts for approximately 90% of diabetes mellitus cases, have been reported to have insulin receptor deletions or mutations. Such deletions and mutations are assumed to cause the disease. Furthermore, some patients with thrombocytopenia have been reported to have thrombopoietin receptor deletions and mutations, and the failure of TPO signaling can be thought to cause the disease. To date, no fundamental therapeutic methods have been available for such genetic diseases, and the establishment of such therapeutic methods is expected.
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that causes thrombocytopenia in infancy and pancytopenia in later childhood. It has been revealed that TPO, a thrombopoietic growth factor, is present in CAMT patients at a high concentration in sera, but that platelets and hematopoietic precursor cells lack TPO responsiveness. Most of these patients have been found to carry point mutations in their thrombopoietin receptor (c-MPL) gene. It has also been reported that such mutations result in frame shift or insertion of a termination codon, leading to patients who have a total loss of thrombopoietin receptor function and patients who have a homozygous or heterozygous amino acid substitution in the extracellular domain of the receptor (see Non-patent Document 1). Bone marrow transplant is the only currently available therapeutic method for treating such patients.
[Non-Patent Document 1]
Matthias Ballmaier, Manuela Germeshausen, Harald Schulze, Klara Cherkaoui, Sabine Lang, Annika Gaudig, Stephanie Krukemeier, Martin Eilers, Gabriele Strausz, and Karl Welte, "BLOOD", vol. 97, No. 1, pp. 139 (Jan. 1, 2001).
The present invention was achieved in view of the circumstances described above. An objective of the present invention is to provide ligands having agonistic activity to mutant receptors.
The present inventors studied intensively to achieve the objective described above. By using antibody engineering techniques, the inventors prepared functional antibodies corresponding to each of the mutations in the causative genes of the diseases described above, discovering that these antibodies were useful to treat such diseases. Specifically, the inventors succeeded in preparing minibodies, each of which has agonistic activity to a mutant receptor that has almost completely lost responsiveness to its natural ligand due to gene mutation (for example, a thrombopoietin (TPO) receptor whose responsiveness to TPO is mostly lost), and which can transduce signals at levels comparable to normal levels when reacted with such a mutant receptor.
Diseases caused by gene mutations can be treated by using such antibodies or modified products thereof. Alternatively, such diseases can be treated by gene therapy using genes capable of expressing the antibodies or modified products thereof.
CAMT patients, who were previously difficult to treat, can now be treated using ligands having agonistic activity, such as the antibodies of the present invention or the genes encoding them.
In addition, the methods of the present invention are applicable to various other diseases caused by mutations in genes encoding receptors on cell membranes. Thus, the present invention can provide new therapeutic methods for these diseases.
Specifically, the present invention relates to ligands having agonistic activity to mutant receptors, more specifically provides:
[1] a ligand having agonistic activity to a mutant receptor;
[2] the ligand of [1], where the ligand is an antibody;
[3] the ligand of [1] or [2], where the ligand has greater agonistic activity to the mutant receptor than the natural ligand;
[4]. the ligand of any one of [1] to [3], where the mutant receptor is a receptor resulting from a mutation(s) in the amino acid sequence;
[5] the ligand of any one of [1] to [4], where the mutant receptor has lost responsiveness to the natural ligand;
[6] the ligand of any one of [1] to [5], where the mutant receptor causes a disease;
[7] the ligand of any one of [1] to [6], where the mutant receptor is a mutant thrombopoietin receptor;
[8] the ligand of [2], where the antibody is a minibody;
[9] the ligand of [8], where the minibody is a diabody;
[10] the ligand of [8], where the minibody is sc(Fv)2;
[11] a method for transducing a signal to a mutant receptor by binding a ligand;
[12] the method of [11], where the ligand is an antibody;
[13] the method of [11] or [12], where the mutant receptor results from an amino acid mutation(s);
[14] the method of any one of [11] to [13], where the mutant receptor has lost responsiveness to the natural ligand;
[15] the method of any one of [11] to [14], where the mutant receptor is associated with disease onset;
[16] the method of any one of [11] to [15], where the mutant receptor is a mutant thrombopoietin receptor;
[17] a method for treating a disease caused by a mutant receptor, by binding a ligand to the mutant receptor;
[18] the method of [17], where the ligand is an antibody;
[19] a method of screening for a ligand having agonistic activity to a mutant receptor, where the method comprises the steps of,
(a) contacting a test substance with the mutant receptor,
(b) detecting a signal in the mutant receptor, and
(c) selecting a ligand having agonistic activity;
[20] a method of screening for a ligand having agonistic activity to a mutant receptor, where the method comprises the steps of,
(a) determining agonistic activity to a normal receptor,
(b) determining agonistic activity to the mutant receptor, and
(c) selecting a ligand having greater agonistic activity to the mutant receptor than the normal receptor;
[21] a method of screening for a ligand having agonistic activity to normal and mutant receptors, where the method comprises the steps of,
(a) determining agonistic activity to the normal receptor,
(b) determining agonistic activity to the mutant receptor, and
(c) selecting a ligand having agonistic activity to both normal and mutant receptors;
[22] the method of any one of [19] to [21], where the ligand is an antibody;
[23] a substance obtained by the method of any one of [19] to [22];
[24] a therapeutic agent for a disease caused by a mutant receptor, where the agent comprises a ligand of the mutant receptor;
[25] the therapeutic agent of [24], where the ligand is the ligand of any one of [1] to[9];
[26] the therapeutic agent of [24], where the ligand is an antibody;
[27] the therapeutic agent of any one of [24] to [26], where the mutant receptor results from an amino acid mutation(s);
[28] the therapeutic agent of any one of [24] to [27], where the mutant receptor has lost responsiveness to the natural ligand;
[29] the therapeutic agent of any one of [24] to [28], where the mutant receptor is a mutant thrombopoietin receptor; and,
[30] the therapeutic agent of any one of [24] to [29], where the disease is congenital amegakaryocytic thrombocytopenia.
The present invention provides ligands having agonistic activity to mutant receptors.
Mutant receptors of the present invention are usually receptors that exist at a frequency of less than 50%, preferably less than 20%, more preferably less than 10%, and even more preferably less than 1%. The frequency is generally calculated using randomly selected subjects. However, the frequency may vary depending on country, area, sex, and such. Therefore, the frequency may also be calculated, for example, within a selected country or area such as Japan, the United States, or Europe, or be calculated for one sex. When there are two or more mutation sites in a receptor, the frequency may be calculated for multiple mutation sites or for any one of the mutation sites. Mutant receptors are preferably evaluated using frequency, as described above. However, mutant receptors can also be evaluated, for example, by their signal transducing ability. Specifically, for example, when two different receptors are present, the one with stronger transducing signals upon natural ligand-binding may be used as a non-mutant receptor, and the other with weaker transducing signals may be used as a mutant receptor.
Preferred mutant receptors of the present invention include, but are not limited to, receptors resulting from amino acid mutations (receptors with mutated amino acid sequences); however, any type of mutation is acceptable, as long as the mutated receptor influences responsiveness to natural ligands, or the conformation, sugar chain structure, or spatial relationship or angles when a receptor exists as a multimer, and so on. Mutations in the amino acid sequence include amino acid substitutions, deletions, insertions, and additions. The receptors of the present invention have preferably lost responsiveness to the natural ligands.
Herein, "ligand" refers to a substance that specifically binds to a functional protein. The type of ligand is not limited. Such ligands include low-molecular-weight compounds, proteins, and peptides. In the present invention, functional proteins are preferably receptors. In the present invention, ligands preferably have agonistic activity. The present invention also provides methods for transducing signals to a mutant receptor by binding a ligand of the present invention. Such ligands for use in the methods of the present invention are preferably non-natural ligands, and not natural ligands.
In the present invention, it is preferred to target a mutant receptor whose responsiveness to a natural ligand is different from that of a non-mutant receptor. A "mutant receptor whose responsiveness to a natural ligand is different from that of a non-mutant receptor" refers to a mutant receptor that exhibits agonistic activity and signaling activity that differs from the activities of the non-mutant receptor when the mutant and non-mutant receptors bind to the same natural ligand under the same conditions. In general, agonistic activity and signaling activity in mutant receptors is impaired compared with non-mutant receptors (the mutants have lost their responsiveness to natural ligands).
The receptors include receptors belonging to the hematopoietic growth factor receptor family, the cytokine receptor family, the tyrosine kinase receptor family, the serine/threonine kinase receptor family, the TNF receptor family, the G protein-coupled receptor family, the GPI-anchored receptor family, the tyrosine phosphatase receptor family, the adhesion factor family, and the hormone receptor family. There are many documents that describe receptors belonging to these receptor families, and their characteristics; for example Cooke B A, King R J B, van der Molen H J Eds. New Comprehensive Biochemistry Vol.18B "Hormones and their Actions Part II" pp. 1-46 (1988) Elsevier Science Publishers BV, New York, USA; Patthy L. (1990) Cell, 61: 13-14; Ullrich A. et al. (1990) Cell, 61: 203-212; Massagul J. (1992) Cell, 69: 1067-1070; Miyajima A. et al. (1992) Annu. Rev. Immunol., 10: 295-331; Taga T. and Kishimoto T. (1992) FASEB J., 7: 3387-3396; Fantl W I. et al. (1993) Annu. Rev. Biochem., 62: 453-481; Smith C A., et al. (1994) Cell, 76: 959-962; Flower D R. (1999) Biochim. Biophys. Acta, 1422: 207-234; Cell Technology: supplementary vol. Handbook series "Handbook for Adhesion factors" M. Miyasaka Ed. (1994) Shujunnsha, Tokyo, Japan, and so on. Specific receptors belonging to the families listed above include: human and mouse erythropoietin (EPO) receptors, human and mouse granulocyte-colony stimulating factor (G-CSF) receptors, human and mouse thrombopoietin (TPO) receptors, human and mouse insulin receptors, human and mouse Flt-3 ligand receptors, human and mouse platelet-derived growth factor (PDGF) receptors, human and mouse interferon (IFN)-α and -β receptors, human and mouse leptin receptors, human and mouse growth hormone (GH) receptors, human and mouse interleukin (IL)-10 receptors, human and mouse insulin-like growth factor (IGF)-I receptors, human and mouse leukemia inhibitory factor (LIF) receptors, and human and mouse ciliary neurotrophic factor (CNTF) receptors (hEPOR: Simon, S. et al. (1990) Blood 76, 31-35; mEPOR: D'Andrea, AD. Et al. (1989) Cell 57, 277-285; hG-CSFR: Fukunaga, R. et al. (1990) Proc. Natl. Acad. Sci. USA. 87, 8702-8706; mG-CSFR: Fukunaga, R. et al. (1990) Cell 61, 341-350; hTPOR: Vigon, I. et al. (1992) 89, 5640-5644; mTPOR: Skoda, R C. Et al. (1993) 12, 2645-2653; hInsR: Ullrich, A. et al. (1985) Nature 313, 756-761; hFlt-3: Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91, 459-463; hPDGFR: Gronwald, R G K. Et al. (1988) Proc. Natl. acad. Sci. USA. 85, 3435-3439; hIFN α/β R: Uze, G. et al. (1990) Cell 60, 225-234, and Novick, D. et al. (1994) Cell 77, 391-400).
In one embodiment, the mutant receptors of the present invention comprise receptors associated with disease onset. The phrase "mutant receptors associated with disease onset" means that the loss of responsiveness to a natural ligand is part of the reason that disease onset is triggered. In the present invention, a mutant receptor is not necessarily the sole factor triggering disease onset, and may be a contributing factor. Many reports have been previously published describing the association of mutant receptors with disease onset; however, in addition to previously reported associations, associations of mutant receptors and disease onset can also be identified by methods of statistical analysis (for example, correlation analyses). Correlation analyses, also called "case control studies", are well known to those skilled in the art (for example, Nishimura, Y., 1991, "Statistical analysis of polymorphisms", Saishin Igaku, 46:909-923; Oka, A. et al., 1990, Hum. Mol. Genetics 8, 2165-2170; Ota, M. et al., 1999, Am. J. Hum. Genet. 64, 1406-1410; Ozawa, A. et al., 1999, Tissue Antigens 53, 263-268). For example, the correlation between a mutant receptor and a disease can be studied by determining the frequency of the mutant receptor in patients and in healthy subjects, and examining whether the patient population has a higher mutant receptor frequency. Typically, differences in the frequency are evaluated using the x-test. x is obtained by the equation x2=Σ(observed value−expected value)2/expected value. A p value is obtained from the x2 value thus determined. Whether a mutant receptor correlates with a disease can be determined based on this p value. For example, when p<0.05, a mutant receptor is considered to correlate with a disease.
There are many reports on mutant receptors known to be involved in disease onset. Such mutant receptors specifically include: mutant thrombopoietin (TPO) receptors, mutant insulin receptors, mutant erythropoietin receptors, mutant growth hormone receptors, mutant common γ chain receptors (common receptor of IL-2, IL-4, IL-7, IL-15, and IL-21), mutant androgen receptors (Glutamine Repeats and Neurodegenerative Disease: Molecular Aspects (2001), 261-267, Oxford University press), mutant receptors for proopiomelanocortin (POMC) and melanocortin (Journal of Clinical Endocrinology and Metabolism (2001), 86(4), 1442-1446), mutant ryanodine receptors (Human Mutation (2000), 15(5), 410-417), mutant thyroid-stimulation hormone receptors (Trends in Endocrinology and Metabolism (1998), 9(4), 133-140), and mutant thyrotropin receptors (European Journal of Medical Research (1996), 1(10), 460-464). In the present invention, particularly preferred mutant receptors are mutant thrombopoietin receptors.
Herein, a "natural ligand" refers to a ligand in the body, and is preferably a ligand with the most influence on non-mutant receptor signaling. Normally, the natural ligands of the present invention do not comprise antibodies.
"Agonistic activity" refers to the activity of inducing a change in a certain physiological activity, caused by transducing signals into cells upon the binding of a ligand to a receptor. The physiological activity includes, but is not limited to, growth-promoting activity, survival activity, differentiation activity, transcription activity, membrane transport activity, binding activity, proteolytic activity, phosphorylation/dephosphorylation activity, oxidation/reduction activity, transfer activity, nucleolytic activity, and dehydration activity.
In the present invention, any detection indicator may be used to assay physiological activities, as long as it can measure quantitative and/or qualitative change. For example, it is possible to use cell-free assay indicators, cell-based assay indicators, tissue-based assay indicators, and in vivo assay indicators. Indicators that can be used in cell-free assays include enzymatic reactions and quantitative and/or qualitative changes in proteins, DNAs, or RNAs. Such enzymatic reactions include amino acid transfers, sugar transfers, dehydrations, dehydrogenations, and substrate cleavages. Alternatively, the followings can be used: protein phosphorylations, dephosphorylations, dimerizations, multimerizations, catabolisations, dissociations and such; and DNA or RNA amplifications, cleavages, and extensions. For example, protein phosphorylation downstream of a signal transduction pathway may be used as a detection indicator. As indicators in cell-based assays, changes to cell phenotype, for example, quantitative and/or qualitative changes in products, changes in proliferation activity, morphological changes, or changes in cellular properties can be used. Products include secretory proteins, surface antigens, intracellular proteins, and mRNAs. Morphological changes include changes in process formation and/or process number, changes in cell flatness, changes in the degree of elongation/horizontal to vertical ratio, changes in cell size, changes in intracellular structure, heterogeneity/homogeneity of cell populations, and changes in cell density. Such morphological alterations can be observed under a microscope. Cellular properties to be used include anchorage dependency, cytokine-dependent responsiveness, hormone dependency, drug resistance, cell motility, cell migration activity, pulsatility, and alteration in intracellular substances. Cell motility includes cell infiltration activity and cell migration activity. Changes in intracellular substances include changes in enzyme activity, mRNA levels, levels of intracellular signaling molecules such as Ca2+ and cAMP, and intracellular protein levels. Furthermore, changes in the cell proliferation activity induced by receptor stimulation can be used as an indicator. The indicators to be used in tissue-based assays include functional changes for the tissue in use. Indicators that can be used for in vivo assays include changes in tissue weight, changes in the blood system (for example, changes in the number of hemocytes, protein contents, or enzyme activities), changes in electrolyte levels, and changes in the circulatory system (for example, changes in blood pressure or heart rate).
The methods for measuring such detection indicators are not particularly limited. For example, luminescence, color development, fluorescence, radioactivity, fluorescence polarization values, surface plasmon resonance signals, time-resolved fluorescence, mass, absorption spectrums, light scattering, and fluorescence resonance energy transfers may be used. These measurement methods are known to those skilled in the art and may be appropriately selected depending on the purpose. For example, absorption spectra can be measured using a conventional photometer, plate reader, or such; luminescence can be measured with a luminometer or such; and fluorescence can be measured with a fluorometer or such. Mass can be determined with a mass spectrometer. Radioactivity can be determined with a measurement device such as a gamma counter, depending on the type of radiation. Fluorescence polarization values can be measured using BEACON (TaKaRa). Surface plasmon resonance signals can be obtained using BIACORE. Time-resolved fluorescence, fluorescence resonance energy transfer, or such can be measured with ARVO or such. Furthermore, a flow cytometer can also be used for measuring. In the present invention, it is possible to use a chimeric receptor comprising an extracellular domain of a mutant receptor and a cytoplasmic domain of another protein. For example, when the cytoplasmic domain of G-CSF receptor, EPO receptor, EGF receptor, or thrombopoietin receptor is used, the cell proliferation activity induced by stimulating the receptor can be used as a detection indicator. In assays using cell proliferation activity as a detection indicator, cell lines that die in the absence of ligands are preferably used to improve detection sensitivity. Cytokine-dependent cell lines are particularly preferred because they can be easily passaged. For example, it is possible to use CTLL-2 cells, which are an IL-2-dependent cell line, and 32D cells, FDC-P1 cells, and Ba/F3 cells, which are IL-3-dependent cell lines. These cell lines will die two or three day after the start of culture when a cytokine such as IL-2 or IL-3 that is essential for cell proliferation, is eliminated from the culture media. It is preferable to use FDC-P1 cells and Ba/F3 cells that express a chimeric receptor comprising the cytoplasmic domain of mouse G-CSF receptor.
The ligands of the present invention that have agonistic activity are not particularly limited, as long as they have agonistic activity to mutant receptors. Such ligands may have agonistic activity to both mutant and non-mutant receptors, or may only have agonistic activity to mutant receptors. When the ligands have agonistic activity to both mutant and non-mutant receptors, they may have greater agonistic activity to the non-mutant receptor, or greater agonistic activity to the mutant receptor. Alternatively, the ligands may have comparable agonistic activity to the non-mutant and mutant receptors. Nonetheless, when the chief purpose is to treat a disease caused by a mutant receptor, it is preferable to use a ligand with greater agonistic activity to the mutant receptor than the non-mutant receptor.
The antibodies of the present invention are not particularly limited, as long as they have agonistic activity, and mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, camel antibodies, chimeric antibodies, humanized antibodies, human antibodies, and such can be appropriately used. Such antibodies may be polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred because they can be stably produced as homogeneous antibodies. Both polyclonal and monoclonal antibodies can be prepared by methods known to those skilled in the art.
Hybridomas producing monoclonal antibodies can basically be prepared by the conventional methods described below. Specifically, immunization is carried out by a conventional immunization method, using a desired antigen or cells expressing the desired antigen as a sensitizing antigen. The prepared immunocytes are fused with known parental cells by a conventional cell fusion method. The fused cells are screened for monoclonal antibody-producing cells (hybridomas) by conventional screening methods.
The type of sensitizing antigen to be used is not limited. For example, a full-length protein of a receptor of interest, or a partial peptide thereof (for example, an extracellular domain) can be used. The antigens can be prepared by methods known to those skilled in the art; for example, according to methods using baculovirus (for example, see WO 98/46777). Hybridomas can be prepared, for example, by the method of Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol., 1981, 73, 3-46.). When an antigen has weak immunogenicity, the antigen may be conjugated with a large immunogenic molecule such as albumin, to achieve immunization. In addition, the present invention may use recombinant antibodies, produced by gene engineering. The genes encoding the antibodies are cloned from hybridomas, inserted into an appropriate vector, and then introduced into a host (see, e.g., Carl, A. K. Borrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies, Published in the United Kingdom by Macmillan Publishers Ltd, 1990). Specifically, using a reverse transcriptase, cDNAs encoding the variable regions (V regions) of the antibodies are synthesized from the mRNAs of the hybridomas. DNAs encoding the variable regions of the antibodies of interest are obtained, and ligated with DNAs encoding desired constant regions (C regions) of the antibodies, and these constructs are inserted into expression vectors. Alternatively, DNAs encoding the variable regions of the antibodies may be inserted into expression vectors comprising the DNAs of the antibody C regions. Those cDNAs are inserted into expression vectors such that the genes are expressed under the regulation of an expression regulatory region, for example, an enhancer and promoter. Host cells are then transformed using the expression vectors, and the antibodies can be expressed. The epitopes on the molecules that are recognized by antibodies of the present invention are not limited to particular epitopes. The antibodies may recognize any epitope on the molecules. Typically, the antibodies recognize epitopes in the extracellular domain. In the present invention, recombinant antibodies artificially modified to reduce heterologous antigenicity against humans can be used. Examples of such recombinant antibodies include chimeric antibodies and humanized antibodies. These modified antibodies can be produced using known methods. A chimeric antibody comprises heavy chain and light chain variable regions of an antibody from a nonhuman mammal such as a mouse, and heavy chain and light chain constant regions of a human antibody. Such an antibody can be obtained by (1) ligating a DNA encoding a variable region of a mouse antibody to a DNA encoding a constant region of a human antibody; (2) inserting the resulting construct into an expression vector; and (3) introducing the vector into a host for production of the antibody. A humanized antibody, which is also called a reshaped human antibody, is obtained by transferring a complementarity determining region (CDR) of an antibody of a nonhuman mammal such as a mouse, to the CDR of a human antibody. Conventional genetic recombination techniques for the preparation of such antibodies are known. Specifically, a DNA sequence designed to ligate a CDR of a mouse antibody with the framework regions (FRs) of a human antibody is synthesized by PCR, using several oligonucleotides constructed to comprise overlapping portions at their ends. A humanized antibody can be obtained by (1) ligating the obtained DNA to a DNA that encodes a human antibody constant region; (2) inserting the resulting construct into an expression vector; and (3) introducing the vector into a host to produce the antibody (see European Patent Application No. EP 239,400, and International Patent Application No. WO 96/02576). Human antibody FRs ligated via the CDR are selected where the CDR forms a favorable antigen-binding site. As necessary, amino acids in the framework region of an antibody variable region may be substituted such that the CDR of a reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856). Methods for obtaining human antibodies are also known. For example, desired human antibodies with antigen-binding activity can be obtained by (1) sensitizing human lymphocytes with antigens of interest or cells expressing antigens of interest in vitro; and (2) fusing the sensitized lymphocytes with human myeloma cells such as U266 (see Japanese Patent Application Kokoku Publication No. (JP-B) H01-59878 (examined, approved Japanese patent application published for opposition)). Alternatively, the desired human antibodies can also be obtained by using antigens of interest to immunize transgenic (Tg) animals comprising a partial or entire repertoire of human antibody genes (see International Patent Application WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735). Furthermore, techniques to obtain human antibodies by panning with a human antibody library are known. For example, the variable regions of human antibodies are expressed as single chain antibodies (scFvs) on the surface of phages, using a phage display method, and the phages that bind to the antigen can be selected. By analyzing the genes of selected phages, the DNA sequences encoding the variable regions of human antibodies that bind to the antigen can be determined. If the DNA sequences of scFvs that bind to the antigen are identified, appropriate expression vectors comprising these sequences can be constructed to obtain human antibodies. Such methods are already well known (see WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388). When the antibody genes are isolated and introduced into appropriate hosts to produce antibodies, hosts and expression vectors can be used in appropriate combinations. Eukaryotic host cells that can be used are animal cells, plant cells, and fungal cells. The animal cells include: (1) mammalian cells such as CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, and Vero cells; (2) amphibian cells such as Xenopus oocytes; or (3) insect cells such as sf9, sf2l, and Tn5. Known plant cells include cells derived from the Nicotiana genus such as Nicotiana tabacum, which can be callus-cultured. Known fungal cells include yeasts such as the Saccharomyces genus, for example Saccharomyces cerevisiae, and filamentous fungi such as the Aspergillus genus, for example Aspergillus niger. Prokaryotic cells can also be used in production systems that utilize bacterial cells. Known bacterial cells include E. coli and Bacillus subtilis. The antibodies can be obtained by introducing the antibody genes of interest into these cells by transformation, and then culturing the transformed cells in vitro.
The antibodies may be minibodies or modified products of antibodies, as long as they can bind to antigens. In the present invention, a minibody comprises an antibody fragment obtained by deleting a portion from a whole antibody (for example, whole IgG). There is no limitation on the type of minibody, as long as it has the ability to bind to an antigen. The antibody fragments of the present invention are not particularly limited, as long as they are portions of whole antibodies. The antibody fragments preferably comprise a heavy chain variable region (VH) or a light chain variable region (VL), and particularly preferably comprise both a VH and VL. Specifically, the antibody fragments include Fab, Fab′, F(ab′)2, Fv, and scFv (single-chain Fv). A preferred antibody fragment is scFv (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883; and Plickthun "The Pharmacology of Monoclonal Antibodies" Vol. 113, Eds. Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)). Such an antibody fragment can be prepared by treating an antibody with an enzyme (for example, papain or pepsin) or by inserting a gene construct encoding the antibody fragment into an expression vector and expressing it in appropriate host cells (see, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; and Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137). The minibodies of the present invention preferably have a smaller molecular weight than whole antibodies. However, the minibodies may form multimers (for example, dimers, trimers, or tetramers), and thus their molecular weights can be greater than those of whole antibodies.
Preferred minibodies of the present invention comprise two or more antibody VHs and two or more antibody VLs, in which each of the variable regions are directly linked, or indirectly linked together via linkers or such. The linkages may be covalent or non-covalent bonds, or comprise both covalent and non-covalent bonds. More preferred minibodies are antibodies comprising two or more VH-VL pairs formed via non-covalent bonding between VH and VL. The distance between the two VH-VL pairs in a minibody is preferably less than that in the whole antibody.
Particularly preferred minibodies of the present invention are diabodies and sc(Fv)2. A diabody is a dimerized fragment in which two variable regions are linked together via a linker or such (for example, scFv) (hereinafter, referred to as "a fragment constituting a diabody"), and which typically comprises two VL and two VH (P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90, 6444-6448 (1993), EP 404097; WO 93/11161; Johnson et al., Methods in Enzymology, 203, 88-98, (1991); Holliger et al., Protein Engineering, 9, 299-305, (1996); Perisic et al., Structure, 2, 1217-1226, (1994); John et al., Protein Engineering, 12(7), 597-604, (1999); Holliger et al., Proc. Natl. Acad. Sci. USA. 90, 6444-6448, (1993); Atwell et al., Mol. Immunol. 33, 1301-1312, (1996)). The links between fragments constituting a diabody may be non-covalent or covalent bonds, and are preferably non-covalent bonds.
Alternatively, two fragments constituting a diabody can be linked together via a linker to form a single-chain diabody (scDiabody). When the fragments constituting the diabody are linked together using a long linker, comprising approximately 20 amino acids, it is possible to link the fragments constituting the diabody in the same chain using a non-covalent bond, forming a dimer.
Fragments constituting diabodies include a VL and VH linked together, a VL and VL linked together, a VH and VH linked together, and the like. A VH and VL linked together is preferred. There is no limitation on the type of linker for linking a variable region and variable region in a fragment constituting a diabody. However, it is preferable to use a linker short enough to prevent formation of a non-covalent bond between variable regions in the same fragment. Those skilled in the art can appropriately select the length of such linkers; however, their length is typically 2 to 14 amino acids, preferably 3 to 9 amino acids, and particularly preferably 4 to 6 amino acids. In these cases, the linker between the VL and VH encoded by the same fragment is short, and thus no non-covalent bonds are formed between VL and VH on the same chain. Thus, a single-chain V region fragment is not formed, and the VL and VH form dimers with other fragments via non-covalent bonds. Further, based on the same principle for producing diabodies, a multimerized antibody such as a trimer or tetramer can be prepared by linking three or more fragments constituting a diabody.
The sc(Fv)2 of the present invention are single-chain minibodies produced by linking two VHs and two VLs with linkers and such (Hudson et al., 1999, J Immunol. Methods 231:177-189). sc(Fv)2 exhibit a particularly high agonistic activity compared to whole antibodies and other minibodies. sc(Fv)2 can be produced, for example, by linking scFv molecules with a linker.
In a preferable antibody, two VHs and two VLs are arranged in the order of VH, VL, VH, and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]), beginning from the N terminus of a single-chain polypeptide.
The order of the two VHs and two VLs is not limited to the above arrangement, and they may be arranged in any order. Examples of arrangements are listed below:
The linkers for linking the variable regions of an antibody can be arbitrary peptide linkers that can be introduced by genetic engineering, or synthetic linkers (for example, see Protein Engineering, 9(3), 299-305, 1996). However, peptide linkers are preferred in the present invention. There are no limitations as to the length of the peptide linkers. The length can be appropriately selected by those skilled in the art, depending on the purpose, and is typically 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and even more preferably 12 to 18 amino acids (for example, 15 amino acids).
For example, such peptide linkers include:
In an embodiment of the present invention, particularly preferable sc(Fv)2 include the sc(Fv)2 below:
Synthetic linkers (chemical cross-linking agents) include cross-linking agents routinely used to cross-link peptides; for example, N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These cross-linking agents are commercially available.
In general, three linkers are required to link four antibody variable regions together. The linkers to be used may be of the same or different types. In the present invention, a preferable minibody is a diabody, even more preferably, an sc(Fv)2. Such a minibody can be prepared by treating an antibody with an enzyme, for example, papain or pepsin, to generate antibody fragments, or by constructing DNAs encoding those antibody fragments and introducing them into expression vectors, followed by expression in an appropriate host cell (see, for example, Co, M. S. et al., 1994, J. Immunol. 152, 2968-2976; Better, M. and Horwitz, A. H., 1989, Methods Enzymol. 178, 476-496; Pluckthun, A. and Skerra, A., 1989, Methods Enzymol. 178, 497-515; Lamoyi, E., 1986, Methods Enzymol. 121, 652-663; Rousseaux, J. et al., 1986, Methods Enzymol. 121, 663-669; Bird, R. E. and Walker, B. W., 1991, Trends Biotechnol. 9, 132-137).
Antibodies with extremely high agonistic activity can be prepared by converting whole antibodies into minibodies.
Modified antibodies for use include antibodies linked to various molecules, such as polyethylene glycol (PEG). Alternatively, it is also possible to link an antibody to a radioisotope, chemotherapeutic agent, or cytotoxic substance such as a bacterial toxin. Such modified antibodies can be prepared by chemically modifying an obtained antibody. Methods for modifying antibodies have been previously established in the art.
Further, antibodies for use in the present invention may be bispecific antibodies. A bispecific antibody may comprise two antigen-binding sites that each recognizes different epitopes on a certain molecule. Alternatively, one of the antigen-binding sites may recognize a certain molecule, and the other may recognize a radioactive substance, chemotherapeutic agent, or cytotoxic substance such as a bacterium-derived toxin. When such cytotoxic substances are used, tumor cell growth can be suppressed by directly adding the cytotoxic substance to cells that express a certain molecule, and specifically damaging the tumor cells. The bispecific antibodies can be prepared by linking pairs of H and L chains from two types of antibodies, or by fusing hybridomas which produce different monoclonal antibodies to produce a fused cell producing a bispecific antibody. Further, bispecific antibodies can be prepared using genetic engineering techniques.
Antibodies in which sugar chains have been modified can also be used in the present invention. Techniques for modifying antibody sugar chains have been previously reported (for example, WO 00/61739 and WO 02/31140).
An "antibody" of the present invention includes the antibodies described above.
The antibodies expressed or produced as described above can be purified by conventional protein purification methods. The antibodies can be separated and purified, for example, by the combined use of methods appropriately selected from affinity columns such as a protein A column, column chromatography, filtration, ultrafiltration, salting out, dialysis, and so on (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
The antigen-binding activity of an antibody can be assayed by conventional methods (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). For example, an enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunofluorescence method can be used.
The present invention also provides methods of screening for ligands having agonistic activity to mutant receptors. In these methods, first, a test substance (test compound) is contacted with a mutant receptor. The "contact" of a test substance with a mutant receptor is typically achieved by adding the test substance to a culture medium or extract of cells expressing the mutant receptor. The methods for achieving contact are not limited to this method. When the test substance is a protein or such, the "contact" can be achieved by introducing a DNA vector expressing the protein into the cells.
In these methods, the next step comprises detecting signals from the mutant receptor. Signals can be detected by the methods described above.
Next, ligands with agonistic activity are selected based on comparison with cases when a test substance (control) is not contacted. The ligands selected in this way are expected to become therapeutic agents for treating or preventing diseases associated with receptor deficiencies or mutations.
In another embodiment, a screening method of the present invention comprises the first step of contacting a test substance with a normal receptor and measuring agonistic activity. Next, the same test substance is contacted with a mutant receptor, and agonistic activity is measured. Ligands with high agonistic activity to the mutant receptor as compared to a normal receptor are then selected.
In still another embodiment, a screening method of the present invention comprises the first step of contacting a test substance with a normal receptor and measuring the agonistic activity. Next, the same test substance is contacted with a mutant receptor and agonistic activity is measured. Ligands with agonistic activity to both the mutant receptor and normal receptor are then selected.
Measuring agonistic activity in the methods described above can be achieved as described above.
Substances (compounds) obtained by the above-described screening methods of the present invention are also comprised in the present invention.
Since the ligands of the present invention (for example, antibodies) have agonistic activity, they are expected to be effective therapeutic agents for diseases caused by the impaired response of receptors on which the ligands act. Such impaired responses are attributed to receptor deficiencies or mutations. Specifically, the present invention provides therapeutic agents comprising the above-described ligands of the present invention, which are used to treat diseases caused by the mutant receptors. Representative examples of the diseases described above are thrombocytopenia, type II diabetes mellitus, and Laron syndrome.
A preferred example of a disease of the present invention is congenital amegakaryocytic thrombocytopenia (CAMT).
When ligands of the present invention or substances (compounds) obtained by the methods for screening of the present invention are used as pharmaceutical compositions, they can be formulated by methods known to those skilled in the art. As necessary, the ligands or substances can be used orally, for example, as sugar-coated tablets, capsules, elixirs, or microcapsules, or parenterally, as injections of sterile solutions or suspensions comprising water or other pharmaceutically acceptable liquids. For example, the ligands or the substances can be formulated by appropriately combining with pharmaceutically acceptable carriers or solvents, specifically, sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents, and such, and mixing at a unit dosage and form required by accepted pharmaceutical implementations. In such formulations, the amount of the active ingredient should be within the required range.
Additives in the tablets or capsules can include, for example, binders such as gelatin, corn starch, gum tragacanth, and gum Arabic; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; edulcorants such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, Gaultheria adenothrix oil, and cherry. When the unit dosage form is a capsule, the above-described materials can also comprise a liquid carrier such as oil. A sterile composition for injection can be formulated using a vehicle such as distilled water used for injection, according to standard protocols.
Aqueous solutions used for injections include physiological saline and isotonic solutions comprising glucose or other adjunctive agents such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. They may also be combined with an appropriate solubilizing agent such as alcohol, specifically, ethanol, polyalcohol such as propylene glycol or polyethylene glycol, or non-ionic detergent such as polysorbate 80™ or HCO-50.
Oil solutions include sesame oils and soybean oils, and can be combined with solubilizing agents such as benzyl benzoate or benzyl alcohol. They may also be formulated with buffers, for example, phosphate buffers or sodium acetate buffers; analgesics, for example, procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or anti-oxidants. The prepared injections are typically aliquoted into appropriate ampules.
The administration may be carried out orally or parenterally, and preferably parenterally. Specifically, injection, intranasal administration, intrapulmonary administration, percutaneous administration, or such can be used. Injections include intravenous injections, intramuscular injections, intraperitoneal injections, and subcutaneous injections. The injection solutions can also be systemically or locally administered. The administration methods can be properly selected according to the patient's age, condition, and such. When the compounds can be encoded by DNA, the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out. The dosage may be, for example, in the range of 0.0001 to 1,000 mg/kg body weight. Alternatively, the dosage may be, for example, in the range of 0.001 to 100,000 mg/person. However, the dosage is not restricted to the values described above. The dosage and administration methods depend on a patient's weight, age, and condition, and can be appropriately selected by those skilled in the art.
Herein below, the present invention will be specifically described using Examples, however, it is not to be construed as being limited thereto.
Several reports have described CAMT patients carrying the G305C (R102P) mutation in their thrombopoietin receptor gene. In this context, an expression vector for the thrombopoietin receptor gene carrying the G305C (RI 02P) mutation was constructed and introduced into Ba/F3 cells by the method described below. The prepared DNA fragments were: the normal thrombopoietin receptor gene (SEQ ID NO: 1) and the mutant gene in which the C at nucleotide position 305 from the initiation codon has been substituted for G (SEQ ID NO: 3). These DNA fragments were digested with the restriction enzymes EcoRI and SalI, and introduced into the EcoRI-SalI site of the animal cell expression vector pCOS2-Ha to prepare pCOS2-hMPLfull (
After the plasmids pCOS2-hMPLfull, pCOS2-hMPLfullG305C, and as a negative control pCOS2-Ha were treated with PvuI, 20 μg of each plasmid was transfected into Ba/F3 cells under the conditions described below. The gene was introduced at a cell density of 1�107 cells/ml in PBS using GENE PULSER II (BIO-RAD) (Gene Pulser Cuvette, 0.4 cm; 0.33 kV; 950 μF). The medium was then changed with RPMI1640 comprising 10% FBS, 1 ng/ml rmIL3 (Pepro tech), 500 μg/ml Geneticin(GIBCO), 100 unit/ml penicillin, and 100 μg/ml streptomycin to select cells. As a result, hMPL-Ba/F3, hMPL(G305C)-Ba/F3, and pCOS2-HA-Ba/F3 cell lines were obtained from the respective vectors described above.
To prepare the antigen for producing anti-thrombopoietin receptor antibodies, a system for producing and secreting the extracellular domain of human thrombopoietin receptor using the insect cell line Sf9 was constructed as described below. A gene construct comprising FLAG tag placed downstream of the extracellular domain of human thrombopoietin receptor (Gln26-Trp491) was prepared and inserted into the PstI-SmaI site of pBACsurf-1 (Novagen), to construct pBACsurf1-hMPL-FLAG (
MRL/lpr mice were immunized seven times with the purified protein of the TPOR extracellular domain. The first immunization was carried out using 100 μg of the protein, and subsequent immunizations were each performed using 50 μg of protein. The immunized cells were fused with P3-X63-Ag8-U1 (P3U1) cells by methods commonly used to prepare hybridomas. The hybridomas that produced anti-thrombopoietin receptor antibodies were selected by ELISA assay using the purified protein (VB08B, VB45B, VB033, VB140, and VB157).
Meanwhile, Balb/c mice were immunized a total of 11 times with hMPL-Ba/F3 cells at one-week to five-month intervals. 1.0�107 cells were intraperitoneally administered to the mice each time. Hybridomas were then prepared by the same method as described above. The hybridomas that produced anti-thrombopoietin receptor antibody were selected (TA136).
The cDNAs for the variable regions of the antibody H and L chains were cloned from each of the hybridomas thus prepared. The cloned cDNAs were sequenced. Based on the nucleotide sequences, genes encoding diabodies were designed with FLAG tag at their C termini (VB08B db, VB45B db, VB033 db, VB140 db, VB157 db, and TA136 db), and inserted into the expression vector pCXND3 for animal cells (pCXND3-VB08B db, pCXND3-VB45B db, pCXND3-VB033 db, pCXND3-VB140 db, pCXND3-VB157 db, pCXND3-TA136 db). Each prepared vector was introduced into COS7 cells, and the culture supernatant was collected after three days of culture. The concentration of diabody in each culture supernatant was determined by BIAcore (Pharmacia) using M2 antibody (Sigma).
pCOS2-HA-Ba/F3 cells, hMPL_Ba/F3 cells, and hMPL(G305C)-Ba/F3 cells were each diluted to 2.0�105 cells/ml with medium (RPMI1640 comprising 10% FBS, 100 unit/ml penicillin, and 100 μg/ml streptomycin). The cells were aliquoted (60 μl/well) into the wells of 96-well plates. hTPO (R&D) was diluted to a final concentration of 25 μg/ml with CHO-S-SFM II, and then aliquoted into the wells (40 μl/well). Each diabody/COS7 sup (VB08B db, VB45B db, VB033 db, VB140 db, VB 157 db, and TA136 db) was diluted 1, 3, 9, 27, 81, and 243 times using CHO-S-SFM II, and then aliquoted into the wells (40 μl/well). The plates were incubated for 24 hours, and then Cell Count Reagent (nacalai tesque) was added to each well (10 μl/well). The O.D.450/655 nm of each well was measured after two hours of culture. The result showed that the responsiveness of hMPL(G305C)-Ba/F3 cells to hTPO and other agonistic antibodies was markedly decreased. However. TA1 36 db (SEQ ID NO: 7) was found to exhibit strong agonistic activity to hMPL(G305C)-Ba/F3 cells, while it exhibited weak agonistic activity to hMPL_Ba/F3 cells expressing the normal receptor (
In SEQ ID NO: 8, the amino acid sequence from positions 49 to 54 corresponds to heavy chain CDR1; from 69 to 84 corresponds to heavy chain CDR2; from 117 to 123 corresponds to heavy chain CDR3; from 163 to 174 corresponds to light chain CDR1; from 190 to 196 corresponds to light chain CDR2; and from 229 to 237 corresponds to light chain CDR3.
As in Example 1, expression vectors were constructed for each of the mutant thrombopoietin receptor genes carrying the C769T (R257C) mutation and the C823A (P275T) mutation, which were found in some CAMT patients. The constructs were introduced into Ba/F3 cells. Nucleotide T was substituted for the nucleotide C at position 769 from the initiation codon in the thrombopoietin receptor gene (SEQ ID NO: 1), to produce the gene of SEQ ID NO: 9; and nucleotide A was substituted for the nucleotide C at position 823, to produce the gene of SEQ ID NO: 11. These DNA fragments were digested with the restriction enzymes EcoRI and SalI, and introduced into the EcoRI-SalI site of the animal cell expression vector pCOS2-Ha to obtain pCOS2-hMPLfullC769T (
After treating the plasmids pCOS2-hMPLfullC769T and pCOS2-hMPLfullC823A with PvuI, 20 μg of each of them was transfected into Ba/F3 cells under the conditions described below. The gene transfer was carried out by electroporation at a cell density of 1�107 cells/ml in PBS using GENE PULSER II (BIO-RAD) (Gene Pulser Cuvette 0.4 cm; 0.33 kV; 950 μF). The medium was then changed to RPMI1640 comprising 10% FBS, 1 ng/ml rmIL3 (Pepro tech), 500 μg/ml Geneticin (GIBCO), 100 units/ml penicillin, and 100 μg/ml streptomycin to select cells. As a result, hMPL(C769T)-Ba/F3 and hMPL(C823A)-Ba/F3 cell lines were respectively obtained from the vectors described above.
TA136 sc(Fv)2 gene was constructed using pCXND3-TA136 db described above by the procedure described below (
PCR was carried out using a combination of primer A (TAGAATTCCACCATGAGAGTGCTGATTCCTTTGTGGCTGTTCACAGCCTTTCCTGGTA CCCTGTCTGATGTGCAGCTGCAGG/SEQ ID NO: 15) and primer B (TGGGTGAGAACAATTTGCGATCCGCCACCACCCGAACCACCACCACCCGAACCACC ACCACCTGAGGAGACGGTGACTGAGG/SEQ ID NO: 16); and also a combination of primer C (CAGTCACCGTCTCCTCAGGTGGTGGTGGTTCGGGTGGTGGTGGTTCGGGTGGTGGC GGATCGCAAATTGTTCTCACCCAGTC/SEQ ID NO: 17) and primer D (ATTGCGGCCGCTTATCACTTATCGTCGTCATCCTTGTAGTCTTTGATTTCCAGCTTGGT G/SEQ ID NO: 18). The resulting PCR products were combined and used as a template in another PCR using primers A and D. The resulting DNA fragment of about 800 bp was digested using the restriction enzymes EcoRI and NotI, and cloned into pBacPAK9 (CLONTECH) to prepare pBacPAK9-scTA136.
PCR was then carried out using pBacPAK9-scTA136 as a template with primer E (GATGTGCAGCTGCAGGAGTCGGGAC/SEQ ID NO: 19) and primer F (CCTGCAGCTGCACATCCGATCCACCGCCTCCCGAACCACCACCACCCGATCCACCAC CTCCTTTGATTTCCAGCTTGGTGC/SEQ ID NO: 20). The resulting DNA fragment of approximately 800 bp was cloned into the pGEM-T Easy vector (Promega).
After confirming the nucleotide sequence, the DNA was digested with the restriction enzyme PvuII. The resulting DNA fragment of approximately 800 bp was inserted into the PvuII site of pBacPAK9-scTA136 to prepare pBacPAK9-TA136 sc(Fv)2. The prepared vector was digested with the restriction enzymes EcoRI and NotI. The resulting DNA fragment of approximately 1600 bp was cloned into the expression vector pCXND3 to prepare pCXND3-TA136 sc(Fv)2 (SEQ ID NO: 13;
The DNA constructs pCXND3-TA136 db and pCXND3-TA136 sc(Fv)2 were introduced into COS7 cells. Their respective culture supernatants were collected after three days of culture. The diabody concentrations in the prepared culture supernatants were determined by BIAcore (Pharmacia) using M2 antibody (Sigma).
hMPL-Ba/F3 cells, hMPL(G305C)-Ba/F3 cells, hMPL(C769T)-Ba/F3 cells, and hMPL(C823A)-Ba/F3 cells were each diluted to 4.0�105cells/ml using medium (RPMI1640 comprising 10% FBS, 100 unit/ml penicillin, and 100 μg/ml streptomycin). The cells were aliquoted into the wells of 96 well plates (60 μl/well). 40 μl of hTPO (R&D) and the culture supernatant of the COS7 cells described above were added to each well, and the plate was incubated for 24 hours. 10 μl of Cell Count Reagent (Nacalai Tesque) was added to each well. O.D.450/655 nm was determined after incubating the plate for two hours.
The results showed that, in all three mutant thrombopoietin receptor cell lines, TA136 sc(Fv)2 exhibited much stronger agonistic activity than hTPO and TA136 db (
The present invention provides ligands (antibodies) to treat patients with diseases caused by mutant receptors, for example, CAMT; polynucleotides encoding these antibodies; vectors comprising the polynucleotides; host cells comprising the vectors; and methods for producing the antibodies. In addition, the present invention also provides methods for gene therapy using polynucleotides that encode the antibodies. The methods of the present invention provide methods for treating various genetic diseases caused by mutations in genes that encode cell membrane proteins. Henceforth, individualized genetic diagnosis is likely to become widely available for patients. The antibody engineering techniques of the present invention enable the development of pharmaceutical agents matched to individual genotypes.
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Hey all, I downloaded some ISOs of older games from ng to backup. Ex. NBA Live 06, NBA 2K6, etc.
For some reason when I mount these using virtual CD I don't see the AUDIO_TS and VIDEO_TS structure like I should, I see nothing. When I burn the discs I get coasters.
I would think it's a problem with that particular ISO but I've seen the same thing three times, all on older games. I can backup newer ISOs just fine.
What is the problem here? Did the ISO format of 360 games change at some point?
Can't Burn Older Isos?
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text:
Associated Press, Published December 31 2009
Insurance license is pulled from agent in Dunn Center, N.D.BISMARCK, N.D. — North Dakota Insurance Commissioner Adam Hamm said he has revoked the insurance license of Patricia Howard, an insurance agent from Dunn Center.
Hamm said she took money and applications from at least two clients but didn't send the documents to the insurance company.
The Insurance Department earlier issued a cease and desist order barring Howard from engaging in the insurance business. Hamm revoked the license after she signed a consent order waiving her right to an administrative hearing.
Copyright © 2009 The Associated Press. All rights reserved. The information contained in the AP News report may not be published, broadcast, rewritten or redistributed without the prior written authority of The Associated Press.


text:
Israel and every politician and statesman interfere at every turn. Say one thing against Israeli policy and you must make a retraction and forever hold your peace. Don't and the Aipac lobby and others will help defeat ANYONE in the next election! Its a well known fact!
If selected for publication, it will appear as soon as possible on Haaretz.com.


text:
FBR Downgrades VMware, Cites 'Fading Tailwinds' From ELAs
FBR Capital Markets & Co. says VMware is in its 'eighth inning' in terms of VSphere penetration in the enterprise, which could limit top-line growth in the future.
Cisco Targets HP, Other SDN/NFV Rivals With Investment In 6Wind
Cisco hopes to take advantage of software from 6Wind that lets industry-standard servers offer SDN and NFV capabilities.
CRN Exclusive: Curvature Taps 35-Year Dell, HP Vet To Boost Channel Strategy
Curvature has tapped Linda York and her 35 years of tech industry leadership experience to boost its network services and channel strategies.
25 Sizzling Gadgets For The Summer
Summer is here, and whether you are lounging by the pool or going hiking with friends, you need one of these 25 cool gadgets to stay refreshed in the hot sun.


text:
Plimoth Plantation is an equal opportunity employer, dedicated to a policy of non-discrimination in employment on any basis including race, color, age, sex, religion, disability or national origin. Consistent with the Americans with Disabilities Act, applicants may request accommodations needed to participate in the application process.
©2003-2015 Plimoth Plantation. All rights reserved.
Plimoth Plantation is a not-for-profit 501 (c)3 organization, supported by admissions, grants, members, volunteers, and generous contributors.


text: Media Google nears launch of paid YouTube subscriptions, report says May 6, 2013 6:47 AM Tom Cheredar 0 If you're not reaching, engaging, and monetizing customers on mobile, you're likely losing them to someone else. Register now for the 8th annual MobileBeat, July 13-14, where the best and brightest will be exploring the latest strategies and tactics in the mobile space. In its quest to pull in premium content, Google is apparently ready to roll out its first set of paid YouTube channel subscriptions. The information comes from a Financial Times report over the weekend that suggests the company could be rolling out paid subscriptions for over 50 different premium channel partners. The subscriptions will reportedly set you back about $2 per month, per channel. We heard rumblings about paid YouTube subscriptions back in January, with the video service rumored to have been in discussions with several of its channel partners about a new subscription-based program. It was also said that those subscriptions would range between $1 and $5 per month, which is in line with the FT report. Google is currently making plenty of money on YouTube videos, but the company realizes it's hitting a brick wall when it comes to gaining the kind of content that people desire from other video services. The company has already spent upwards of $200 million to market and cultivate premium content through its channel partners, but so far it's still not lucrative enough for those partners. The subscription would likely be another source of revenue both for Google and those content creators. We're reaching out to YouTube for further comment about the paid channel subscriptions and will update this post with any new information. Via CNET; Photo via Korosirego/Flickr VB's research team is studying mobile user acquisition... Chime in here, and we'll share the results.


text:
Hacking North by NorthEast...
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Deaf on Wheels
Joined: Dec 2007
Location: Prescott, Arizona, USA
Oddometer: 2,310
On the way to the "ROCK"
Arrived cold at the terminal for our sail on the M/V Caribou to Newfoundland-"The Rock"
Submitting papers..tickets..and OH, they forgot to add the sidecar to the bill of lading...so another $72 for my contraption..
Lotsa $$$$....2 people; bike; sidecar one way to Port aux Basques..7hrs
Leave Argentia, NFLD to North Sidney, Nov Scotia..14 hr; with cabin for 2
Well worth it...
Met a couple from Texas on their Honda GW and .....
They have done most corners but have yet to do Alaska and Labrador. Going only on the last paved Lab road to Red Bay and return..
Taking some rays...pretty cold here...While waiting to bord..
A very large complement of Trucks, cars, Campers, etc but only 2 motorcycles...
Ready to get swallowed..
Upper Deck...
These Lady crews were so enmored with our rig that they asked if they could sit in it and be pictured...lovely Gals. WOuld have given them a ride if allowed on deck...
Aboard Ship this is where all transactions, matters ship are done..
Model of our ship..
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text: It's Limp Bizkit Frontman Fred Durst's Yearbook Photo! By Liz Ramanand July 18, 2012 6:48 PM Share on Facebook Share on Twitter Michael Buckner, Getty Images Sorry, you need to have javascript running to see this poll. Next Rock Yearbook Photo Category: Special Features Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! Recommended For You Around the Web Best of Loudwire Rob Zombie Fans Play Mad Libs Karaoke at the 2015 Loudwire Music Festival 100 Metal Facts You May Not Know This Dude on the Subway Could Be an Extreme Metal Vocalist Lamb of God's Randy Blythe Talks Quitting Alcohol, Perception of His Czech Arrest + More


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Sale heads list of potential Sox ASG reserves
Historic strikeout run highlight's lefty's dominance
CHICAGO -- Chris Sale figures to be selected as an All-Star for the fourth straight year when reserves are announced Monday night. The left-handed starter also seems to be the only viable White Sox candidate, unless first baseman Jose Abreu makes his way onto the Final Vote ballot.
With double-digit strikeouts recorded in eight consecutive games, joining Pedro Martinez as the only Major Leaguers to accomplish such a feat, Sale has drawn increased notice from those outside of Chicago and the Midwest. And he already was considered one of the game's best starting pitchers before this run.
Scott Merkin is a reporter for MLB.com. Read his blog, Merk's Works, follow him on Twitter @scottmerkin and listen to his podcast. This story was not subject to the approval of Major League Baseball or its clubs.


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Truckin Magazine
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Top StoryFord Transit Sets Sales Record for June 2015
Replacing the E-Series/Econoline van with the unibody Transit was a huge gamble for Ford, but if its first year of sales is any indication, it paid off.
Latest Truck Industry News Articles
June 2015 SUV Sales—Compact Crossovers Dominate
SUV sales for June 2015 trended distinctly toward compact crossovers, as usual. The Honda CR-V, Toyota RAV4, and Ford Escape rounded out the podium finish.
June 2015 Truck Sales — GM Flexing Truck Muscles, Toyota Strong
GM Claims Silverado Retail Sales Ahead of F-150, Colorado Lowest Turn Rate
Hyundai Creta Hints At Future Crossover for U.S.
Global Versions Offer Diesel Option, Premium Features
Euro SUV Rumor Roundup: Macan-Fighting Lotus, Smaller Bentley
Lotus Says SUV Due in 2019 Will Be "Better" Than Macan
New Hennessey VelociRaptor SUV Moves to F-250 Bones
Hennessey Performance presents an alternative to the aged Excursion, the new 2015-2016 VelociRaptor SUV. Based on the current F-250, it's perfect for big jobs.


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Viewing 1 — 25 of 2314 results matching your search critieria in Production Assistants in Huntington Beach, California
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Our mission is to provide outdoor recreational activities for Disabled American Veterans, disabled individuals and disabled youth in Minnesota. Because of their disabilities, the individuals and groups that we help dont have enough funds, volunteers or help to do these things. Often times these handicapped people, who are family, friends and neighbors, do not have the opportunity to do many of the activities that others enjoy. Our goal is to help them to enjoy these actives that people who dont have disabilities enjoy and to improve their quality of life, to help them feel they are not forgotten. Be it lack of funds or volunteers or the handicap accessible places to enjoy the outdoor recreation that others do.
You Can Help us Change Lives
People who live with disabilities are often reminded of their limitations. Would you like to help them turn that around, and remind them of what recreational opportunities they are truly capable of?
We provide safe, guided, hands-on outdoor experiences. We give friends, family, and the entire community a way to participate with those with disabilities in an open friendly way.
Help us Build Community
It's people just like you, the local folks, the local community that helps Midwest Outdoors Unlimited reach out to those with disabilities and help get them outdoors. You are welcome to get involved.
Midwest Outdoors Unlimited 28791 325th Ave., Melrose, MN 56352 Phone: 320.260.6023 firstname.lastname@example.com
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text: The Branded wiki last edited by Bogitt on 10/24/13 11:29PM View full history OverviewThe Branded are a shunned race of people seen in the Tellius Fire Emblem universe of Fire Emblem: Path of Radiance and Fire Emblem: Radiant Dawn. They are the mixed-blood offspring of beorc/ laguz parentage and are so named due to bearing a brand on their bodies that marks them as neither fully beorc nor laguz.Stefan is a powerful Branded warrior that lives in a secret colony where his kind live in peace.Branded are otherwise physically indistinct from a pure-blood beorc, as they lack the bestial traits of their laguz parents. However, once they reach a certain age, their aging process slows down dramatically, giving them a laguz's life expectancy. Due to this trait of their aging, it is impossible for them to remain hidden in a beorc society for more than a few years.Laguz are able to sense the presence of Branded as a sort of sixth sense. Though they are not outwardly hostile to Branded, their common behavior when one is in their midst is to treat the Branded as if he or she does not exist. Laguz that either father or give birth to a Branded child lose their powers, and thus Branded are seen as abominations that the laguz refer to as the Parentless.Branded vs. Spirit CharmersAlmedha gave birth to a baby fathered by the beorc Ashnard, and as a result lost her powers.Branded are sometimes confused for spirit charmers, or beorc that make pacts with powerful spirits in return for great magical power. This is due to the fact that spirit charmers also bear brands on their bodies as a sign of the pacts that they have made. Pelleas is one such spirit charmer, and it is because of the brand on his body that Izuka is able to trick the people of Daein into believing that the young man is in fact the lost son of King Ashnard and his laguz lover, Almedha.Conversely, the character Soren was an orphan raised at one point by an old man under the mistaken belief that he was a spirit charmer due to the mark on his forehead. In fact, Soren is a Branded, and if the proper prerequisites are met at the end of Radiant Dawn, it is revealed that he is in fact the true son of Ashnard and Almedha.Ashera's JudgmentIn Fire Emblem: Radiant Dawn, Ashera judges the beorc and laguz both unworthy for their continued acts of warfare and uses her powers to turn all to stone. At first, it is believed that only those powerful enough to resist her judgment survived. However, when Amy, the adopted child of Largo and Calill is brought to the camp safe and sound, it is revealed that the judgment had no effect on the Branded.


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Ickenham, St Giles' Church c.1965
This is the 2nd of 6 photos of Ickenham.
Memories of Ickenham
12 Glebe Avenue Kolordek
This picture is just too small to see if my parents' shop - Kolordek - is illustrated in the row. We moved away around 66/67. Vaiseys had the grocers next door - I was friends with their daughter, and the grocer's ...Read full memory
Glebe Avenue Parade Of Shops
..or 1959 to 1964 The other grocers on that parade may have been the DeHond family, I think that they originated in Belgium? A very pleasant, kindly, hardworking family. I used to help out by driving their ...Read full memory
St Giles Church.
We moved to Ickenham in early 1960s and attended the parish church of St Giles. It is a very old church, once belonging to the estate of Swakeleys House, and is on the corner of a very busy T junction, but when inside it is ...Read full memory
Shopping At Hamers
I moved to St Giles Avenue with my parents in 1948 when I was 15 years old. Before this group of shops was built, there was a little hut run by Mr Hamer and I remember my dad buying his cigarettes and newspapers there. ...Read full memory
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Wood, P; Caldwell, DE; Evans, E; Jones, M; Korber, DR; Wolfhaardt, GM; ... Gilbert, P; + view all Wood, P; Caldwell, DE; Evans, E; Jones, M; Korber, DR; Wolfhaardt, GM; Wilson, M; Gilbert, P; - view fewer (1998) Surface-catalysed disinfection of thick Pseudomonas aeruginosa biofilms. J APPL MICROBIOL , 84 (6) 1092 - 1098.
Full text not available from this repository.
Transition metal catalysts were incorporated into polymers which formed the surface for bacterial attachment and biofilm formation in a constant depth film fermenter (100 mu m thickness), flow chamber (about 30 mu m thickness) and in batch culture (< 30 mu m thickness). The catalysts drive the breakdown of persulphates to reactive oxygen species. When Pseudomonas aeruginosa biofilms were exposed to dilute solutions of potassium monopersulphate (20 mu g ml(-1)-1 mg ml(-1)), significant enhancement of killing was notable for catalyst-containing surfaces over that of controls. The degree of enhancement was greatest for thin films, but was nevertheless significant for the 100 mu m thick biofilms. Fluorescence probes and viability staining, in conjunction with laser confocal microscopy, showed that reactive species were generated at the biofilm-substratum interface and killed the biofilm from the inside. Reaction-diffusion limitation now concentrates the active species within the biofilm rather than protecting it, and a diffusion pump is established whereby further treatment agent is drawn to the substratum enabling relatively thick biofilms to be disinfected.
|Surface-catalysed disinfection of thick Pseudomonas aeruginosa biofilms
|UCL classification:
|UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Eastman Dental Institute > Microbial Diseases
Archive Staff Only: edit this record


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MRM 2 years, 11 months ago
TSPLOST is a net loser for SWGA. The projects listed are just those proposed. The final list will be approved by an unelected board. There is no way that SWGA gets enough money from TSPLOST to do all the projects on the list. And any new roads, widening projects, etc have to be maintained. Where does that money come from.
Transportation infrastructure should be paid by user fees and user taxes, not a general sales tax. This hurts the poor the most cause they will benefit from it the least. The original MARTA SPLOST was supposed to fix all of MARTA's problems. MARTA is still broke.
We don't need new taxes, we need jobs which comes from having lower taxes, government regulations, and attracting PRIVATE capital into the area. This tax does exactly the opposite.
Also there is the constitutionality of the issue. There is no provision in the Georgia Constitution for a regional sales tax - only a local and a statewide sales tax. What about the counties that vote down the tax yet the region approves it??? How is that fair to FORCE them to comply?
This tax does not create jobs. It just shifts them around because all this does is shift money from one person's pocket to another. Yes we may get a few more road construction jobs. But we will lose retail jobs as people can afford to buy less at the retail level as government takes another slice out of their declining incomes.
This tax is a rotten egg. IT STINKS!!!


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3 bed, 2 full bath. Lots of improvements - hardwood floors in Great Room 7 Master BR, hall bath completely redone - MBRMore info
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Tuesday's boys volleyball roundup
BOYS' volleyball
Ventura 3, Buena 0
(Scores: 25-16, 25-17, 25-13)
At Ventura
Ventura: Dustan Neary had eight kills and four blocks. Aiden Fenwick had six kills and eight digs. Natha Adair also had eight digs and added six aces. Zach Svenson had 24 assists.
Buena: Hayes Martin had six kills, hitting .500, with three blocks. Nate Grates had four kills and four blocks. Spencer Smith had six kills. Parker Jellison added four kills and two blocks. Karlis Tolks had 19 assists and four digs.
Records: Ventura 9-3, 6-3 Channel League; Buena 2-9, 0-3.
(Scores: 25-19, 25-21, 25-22)
At Rio Mesa
Rio Mesa: Zech Burkhardt had 10 kills and five digs. Paul Dubuke had 12 kills. Brett McKinney had eight kills and seven blocks.
Record: Rio Mesa 10-5, 3-0 Pacific View League.
(Scores: 25-21, 25-18, 26-24)
At Oxnard
Oxnard: Chris Bossoletti had 33 assists. Ed Rodriguez had 12 kills and six digs. Nathan Sanchez had 10 kills.
Camarillo: Connor Hoybjerg had 10 kills, Danny Reilly had eight kills and Jesse Flitt had 24 assists.
Records: Oxnard 5-5, 2-1 Pacific View League; Camarillo 11-4, 2-1.
Hueneme 3, Channel Islands 2
(Scores: 25-21, 18-25, 16-25, 26-24, 15-8)
At Channel Islands
Hueneme: Patrick Moeslein had 18 kills, 10 blocks and six digs. Ben Caluya had nine kills and 10 digs. A.J. Cabardo had 36 assists.
Channel Islands: Miah Balowaga had 16 kills and 12 blocks. Sean Simeon had 17 kills. Tyler Coleman had eight kills and three blocks.
Records: Hueneme 2-4, 1-2 Pacific View League; Channel Islands 5-11, 0-3.
(Scores: 25-12, 25-12, 25-12)
At Fillmore
Fillmore: Chris De La Paz had nine kills and 11 digs. Nick Paz had nine assists.
Record: Fillmore 0-13, 0-8 Tri-Valley League.
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Deric Longden Message Board
Post to this discussion forum
Return to Deric Longden book reviews
Robin and Maz
posts on 6/23/2013 5:35:17 PM
Thank you Annie for letting us all know this terribly sad news. We do hope that it is some consolation to you to know how much Deric was loved by so many and will continue to do so. He gave us all a wonderful gift in his books and musings and we will continue to laugh and cry as we read them again and again. Thanks also for making that easier with Deric's website and Kindle books. Our thoughts and wishes go to Aileen, all Deric's and Aileen's children including yourself and grandchildren Bless you all Maz and Robin X
Jenny Reeve
posts on 6/23/2013 5:23:21 PM
Dear Annie. I am sure I speak on behalf of us all here and also fans of Deric the world over, I offer my deepest sympathies to you and your family. What a pleasure it has been and will always be to read the wonderful writing that only Deric Longden could write so well. He will be so dearly missed. Our thoughts are with you all. God bless xx
Sue Wallace
posts on 6/23/2013 5:21:04 PM
Oh Annie, this is the news that I think we've all been dreading but thank you so much for posting it at what must be such a sad time for you all. I am very, very sorry to hear that Deric has passed away and send lots of love to you all. He will always remain in my heart for as long as I draw breath. XXXX
Annie Q
posts on 6/23/2013 5:05:22 PM
Sorry to have to tell you that Deric passed away this morning after a brave battle with throat cancer. We are here with Mum now and the family is in deep shock and rallying around. Deric's words will forever live on in his wonderful books and his love of life and laughter has blessed us all. Thank you for your loyalty and superb support which has always given Deric so much pleasure.
Yvonne Welsh
posts on 6/22/2013 3:07:06 PM
Hi all, been a long time since I posted here! Hope everyone is doing well. Since my last post I have gained a kitten! Elsa wasn't too pleased the first two days but they both soon started playing together. I'm having to get used to the rough and tumble play fighting- never had two cats before so new for me too!We have named the kitten Simmi, short for Simoncelli, a favourite motorbike rider of ours who died two years ago. Simmi will be 10 weeks old tomorrow and has settled here well. She loves Keiths lap to sleep on and is there as I type! Hugs to all! x x
posts on 6/20/2013 8:30:14 PM
There is a cute little story about Deric on the examiner dot co dot uk site.by Denis Kilcommons. You have to do a search for it at the site since I can't post the web page here.
posts on 6/20/2013 7:56:42 PM
Hi Gill, has been a while since I posted and it appears to be a while since anyone has posted. I am really disappointed that it has taken 13 years for you to get a diagnoses for your son. We received ours when Daniel was nearly 9 and that was considered late! I can really appreciate the horror that your family and son has gone through, it is not easy having an austist as a child, but there are great rewards too. After coming into contact with the subject, I too was amazed at how many people are autistic, so I have come to the conclusion that they are in the majority. What a shame they don't run the country, would be a better place to live in I'm sure. Greetings to everyone.
posts on 6/20/2013 5:59:32 PM
Hi Jan, and sugeebas, and all who post.. Thank you for your lovely kind words. I am always surprised by just how many kids there are out there with autism. My son is nearly 13, and has struggled most of his life with school, and social situations, so it was a relief to finally get a diagnosis, of course no-one tells you that is just the beginning of a insurmountable mountain! As for my dear beautiful friend, I miss her every day. We had known each other 26yrs, and she was the sister I never had...I still cannot believe she has gone, i know I will never have a friendship like that again in my lifetime, so try to be thankful that I was lucky enough to have shared her with her family and friends for as long as I did. Deric and Aileen, still in my thoughts...recommend your books to customers frequently as it seems lovers of coffee and tea are lovers of cats too! Enjoy the weekend everyone..what's everyone up too? Gill, belper.xx
heather dunn
posts on 5/27/2013 2:54:11 PM
hello all hope everyone is well and all the pussies. One of mine is just recovering from a fractured pelvis. Wehad to confine her to a dog crate for 2 weeks but she coped well, I think she quite liked her own little space!
posts on 5/27/2013 11:39:11 AM
Hi Everyone! Jocelyn, I was SO sorry to hear about Tandy. It's very hard when a beloved pet dies as they do become part of your family. However I'm glad you have her kitten to soften the blow a little. Our first rescued cat in 2001 was only with us 8 months as it was discovered she had cancer on her tongue. I'd put so much effort into that timid little cat and she'd become a lovely lapcat. I surprised myself at how upset I was! Jan, we didn't go on the QE2 as that was sold some years ago - we went on the 3 year old Queen Elizabeth, named by our current Queen here in Southampton. She is the smallest of the '3 Queen ships' and is absolutely beautiful. Deric, we haven't heard anything of you, nor do we expect to, but I do hope you and Aileen are keeping as well as you can, especially in this beautiful sunshine that has been with us this past week - hopefully summer will come! Love to all, Diana - Southampton xx
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4.55 ct. t.w. CZ Station Necklace in Sterling Silver. 18"
Item#: 830580
You'll reach for this versatile necklace again and again. Stations of pear-shaped CZs, 3.00 ct. t.w., are interspersed with round CZ clusters, providing just the right amount of sparkle for every day. Cable chain, lobster clasp. Sterling silver necklace. CZ weights are diamond equivalents.
Due to the incredible value on your Clearance selection, no additional discounts apply.
Product Details (for 4.55 ct. t.w. CZ Station Necklace in Sterling Silver. 18")
5/16 inch
Sterling close STERLING SILVER: Also known as "Standard Silver," sterling silver is silver alloyed with other metals to increase the hardness and durability. Generally, sterling is 92.5% silver and 7.5% other metals. Some sterling silver jewelry is plated with 14kt-18kt gold and referred to as "vermeil," or "gold over sterling." Silver
Polished close POLISHED: A buffed-out finish used to produce a mirror-like shine.
Cable close CABLE: One of the most popular and classic of chain styles, created from interlocking oval links unvarying in size.
Chain Size:
1.5 mm
Lobster close LOBSTER CLASP: named after the pinching style of the hook, this clasp is often shaped like a lobster's claw. Use your finger to work the lever to open the clasp.
Gemstone 1:
Cubic Zirconia close CUBIC ZIRCONIA: once called the poor cousin to the diamond, CZs have since become a fashion statement in their own right. Perfect for travel and a little extra bling, CZ Jewelry is one of man's flawless creations. (Color: Colorless, Quantity: 4, Shape & Cutting Style: Pear close PEAR: Similar to the outline of a pear fruit, this is a round shape that comes to a point on one side. , Weight: 3.00 ct. t.w., Size: 7X5 mm)
Gemstone 2:
Cubic Zirconia (Color: Colorless, Quantity: 3, Shape & Cutting Style: Round Brilliant close BRILLIANT: A gemstone cut to enhance brilliance without sacrificing much carat weight. Typically has 58 facets. , Weight: 0.75 ct. t.w., Size: 4 mm)
Gemstone 3:
Cubic Zirconia (Color: Colorless, Quantity: 88, Shape & Cutting Style: Round, Weight: 0.80 ct. t.w., Size: 1 mm)
All weights and measures are approximate.
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Headrest Printer Mounts - LEM Solutions
LEM Solutions has solved the "where do you put it" question with its patented...
The measure would leave police officers who violate the policy open to lawsuits if they are found to have acted in bad faith or gross negligence.
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Bio: For years, Upon Graves was merely an idea that lay dormant in the mind of drummer and lyricist, Ian "Mortem" Antonelli. However, it wasn't until 2012 that the idea would manifest into something greater. Having previously jammed with Justice Owens, he knew it was the right choice to join forces ag...See Full Bio
Prepare yourselves, for we shall be unleashing new music. The recording of our EP "The Devil's Hellfire" begins next week.


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Toscanini's Ice Cream Company (known simply as Toscanini's or Tosci's) is an ice cream parlor and café in Cambridge, Massachusetts, founded in 1981. It has won the Best of Boston award for best ice cream in 1997,[1] 2009,[2] and 2010,[3] as well as other Best of Boston awards.[4] It has also been highly rated in Gourmet magazine,[1] and claims to have been highly rated in the New York Times food section.[5]
Toscanini's original (and presently only) location is at 899 Main St. in Central Square. Other locations have included the Someday Café in Somerville, Massachusetts (closed August 2006), a location in the MIT student center (closed December 2000), and a location on Massachusetts Avenue in Harvard Square (closed December 2006). Toscanini's ice cream can also be found in some restaurants and supermarkets in the Boston area, such as Whole Foods Market.
Owner Gus Rancatore recently published an Amazon Short with author Helen Epstein, entitled Ice Cream Man: 25 Years at Toscanini's,[6][7] presenting a personal story of how Toscanini's came to be. The short has spent several weeks on Amazon's top ten list.
Seizure by the Massachusetts DoR[edit]
On January 17, 2008 the business was seized by the Massachusetts Department of Revenue because of failure to pay more than $167,000 in taxes dating back to 2000. Ice cream maker Samuel Mehr set up a website with a PayPal donation link,[8] and an overwhelming amount of support from the local community and beyond provided the owners with more than $30,000 toward satisfying the tax bill. A payment plan was organized with the MA DOR to pay the balance. After being closed for eight days, Toscanini's Ice Cream and Coffee Shop re-opened for business on January 25.[9]
- ^ a b Tosci's Ice Cream Wins Boston Award
- ^ Best of Boston 2009 Ice Cream: Toscanini's
- ^ Best of Boston 2010: Ice Cream Toscanini's
- ^ Search Results: toscanini's
- ^ About Toscanini's, from Toscanini's official web site
- ^ Ice Cream Man: 25 Years at Toscanini's, an Amazon Short by owner Gus Rancatore with Helen Epstein
- ^ Ice cream maker still sweet on MIT: Toscanini's owner looks back on 25 years, an article in MIT Tech Talk
- ^ Goodnough, Abby (January 30, 2008). "Special Flavor: The Big Rhubarb". The New York Times. Retrieved May 25, 2010.
- ^ Abelson, Jenn (January 26, 2008). "Shop is set to lick its tax problem". The Boston Globe.
External links[edit]
Coordinates: 42°21′48.6″N 71°5′57.5″W / 42.363500°N 71.099306°W


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FIG. 1 is a front view of a set of key buttons for a handset showing my new design; and,
FIG. 2 is a perspective front view thereof.
The broken line showing of a remainder of a handset in the figures is for illustrative purposes only and forms no part of the claimed design.