text:
Doodle God™
866 reviews
By installing you agree to the Terms of Use and other terms
PublisherMicrosoft Studios
More from Microsoft Studios
Download size24 MB
Content rating
Last updated11/18/2011
Works with
- Windows Phone 8.1
- Windows Phone 8
- Windows Phone 7.5
App requires
- Xbox
- phone identity
- photo, music, and video libraries
- data services
- HD720P (720x1280)
- WVGA (480x800)
- WXGA (768x1280)
*Not available for all phones Learn more
Supported languages (1)
Over 55,000,000 players already tried to create their own world in award-winning fantastically addictive worldwide hit Doodle God™! ******* What's inside your Windows Phone? Circuits, microchips, batteries? No! Your WP is full of fire, water, earth and air! What do you do with your phone? Make calls, send texts, check emails? Boring! With Doodle God™ you can create storms, build armies, grow a civilization! Doodle God™ puts the power of creation in your hands. Mixing and matching different elements, work your way up, all the way from bacteria and beetles, to clay and ceramics, to tools, weapons and beyond! • Mold the 4 basic elements to your whim. • Create 400+ advanced items including exclusive WP episodes. • Intuitive one-click gameplay encourages thoughtful, creative play. • Xbox LIVE features, Leaderboards and Achievements. • Hundreds of interesting, funny and thought-provoking quotes and sayings. • Mini-Games section for arcade fans. "…you realize the true genius behind Doodle God's alluring gameplay", "If you're open to a new gameplay experience … I recommend giving Doodle God a whirl." – TouchArcade.com "Wonderfully unique concept. Deeply satisfying every time you discover a new element." – GameZebo.com ******* Follow us to get early access to exclusive content and updates: www.facebook.com/doodlegod www.twitter.com/#!/joybitsmobile
6/29/2015One of the best games in the store
6/6/2015This game kicks ***
6/6/2015Please please please please please make it free :) because I love this game please please please please please
6/3/2015I LOVE💗this game. THANKS
5/12/2015Make it free for 1 day
4/27/2015Zomg its terrible,,,,,
4/1/2015Hours of fun
3/4/2015It's Pretty Fun I like It A lot
3/1/2015I love ❤ you


text:
The mission of the Junior Volleyball Association is to promote the growth of youth and junior volleyball
through program and resource development, education and events.
This JVA award honors junior beach players for outstanding achievement in beach volleyball this season. Click here to submit your nominations.
48 girls junior volleyball players are honored for their outstanding achievement on the volleyball court as the top JVA players in the country. Click here to view the honorees.
18 student-athletes are named JVA AthLeaders for the 2015 season for their commitment to excellence outside of volleyball through academics, leadership, extra-curriculars, and community service. Click here to view the honorees.
The Beach U Tour commences in the Midwest!Register your teams today! Click here for more informatiion.
The Official Beach Sock of the JVA

subset: commoncrawl


text:
Embodiments of the invention relate to a method for treating target cells, tissues or pathogens in a subject, comprising administering a non-covalently bound complex which comprises a multispecific targeting protein and a hapten-enzyme covalent conjugate, followed by administration of a prodrug that is converted to an active drug by the enzyme. Other embodiments of the invention further relate to kits comprising the non-covalently bound complex or the components to prepare the complex.
1. A method for treating target cells, tissues or pathogens in a subject comprising:
a) mixing
(i) a multispecific targeting protein comprising at least one target-binding site and one hapten-binding site, and
(ii) a hapten-enzyme covalent conjugate comprising two haptens linked by a peptide of from 2 to 10 amino acid residues in length, wherein the hapten-enzyme covalent conjugate is a carboxylesterase-IMP222 (Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH2 (SEQ ID NO: 2));
b) forming a non-covalently bound complex between the multispecific targeting protein and the hapten-enzyme covalent conjugate, wherein the non-covalently bound complex is formed prior to administration of the complex to a subject;
c) administering the non-covalently bound complex to a subject; and
d) administering to the subject a chemotherapeutic drug or prodrug, capable of being converted to a more active drug by the enzyme attached to the non-covalently bound complex.
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
11. The method of
12. The method of
13. The method 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 method of
21. The method of
22. The method of
23. The method of
This application is a continuation-in-part of application Ser. No. 10/714,391 (now abandoned), filed Nov. 17, 2003, which claims priority from provisional application Ser. No. 60/426,379, filed Nov. 15, 2002; and is also a continuation-in-part of application Ser. No. 11/198,846, filed Aug. 8, 2005, now U.S. Pat. No. 7,560,110 which is a divisional of application Ser. No. 10/150,654 , filed May 17, 2002, now U.S. Pat. No. 7,138,103, which is a continuation-in-part of application Ser. No. 09/382,186, filed Aug. 23, 1999, now U.S. Pat. No. 7,052,872, which is a continuation-in-part of application Ser. No. 09/337,756, filed Jun. 22, 1999, now U.S. Pat. No. 7,074,405, which claims priority from provisional application Ser. No. 60/104,156, filed Oct. 14, 1998 and provisional application Ser. No. 60/090,142, filed Jun. 22, 1998.
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 20, 2010, is named 78258341.txt, and is 9,660 bytes in size.
The invention relates to a method, a composition and a kit for delivering therapeutic agents to subjects.
The selective delivery of therapeutic agents to diseased tissue, in vivo, remains a major challenge in the interests of improved therapeutic outcomes. It must be appreciated that although much of the following discussion of the invention is specified toward anti-cancer treatments, any disease state amenable to treatment with drugs or prodrugs could be addressed in the same way. In the case of cancers, standard chemotherapies have depended, in general, upon the enhanced uptake of toxic drugs by fast-growing diseased cells, in relation to most normal cells. However, this has been found to be only of limited value, and normal cell toxicities are often reached before a decisive therapeutic effect against the cancer can be obtained. Typically, those normal cells that divide the fastest are most prone to the adverse effects of chemotherapy agents.
Several different approaches are being taken that seek to improve the therapeutic outcomes resulting from anti-cancer drug therapies. One is the use of mixtures or 'cocktails' of drugs, with component drugs often chosen for their effects on different aspects of cell metabolism. A second is the encapsulation of drugs into carriers such as liposomes or the attachment of drugs to long-circulating polymers. This approach extends drug half-life in serum, and generally allows for a greater proportion of the administered drug to be deposited at the target site. A third approach can be viewed as an advance on the second approach, in that drugs are attached to specific targeting agents such as monoclonal antibodies or peptides. These agents are able to specifically accrete at a target due to their binding to an antigen or receptor, respectively, which has been upregulated or specifically produced by the target cells.
A disadvantage with the aforementioned approaches is the tendency of drugs to lose potency upon conjugation to a polymer, peptide or monoclonal antibody (MAb). Numerous articles have described methods of drug conjugation that seek to preserve drug activity while forming a stable bio-conjugate. Unfortunately many drug-carrier conjugates also dissociate when subjected to the challenge of an in vivo serum environment. Moreover, tumor uptake of the drug is often reduced while non-specific toxicity to normal tissues is often increased.
An advanced method for delivering a drug to a disease site in a less toxic and more efficient and efficacious manner, is the use of antibody-directed enzyme prodrug therapy (ADEPT). See, for example, U.S. Pat. No. 5,632,990 (Bagshawe). Originally, ADEPT depended on the use of a conjugate of an antibody and an enzyme to localize the latter to a site of disease. Such an approach had several drawbacks, including loss of antibody and/or enzyme activity upon conjugation, and high residual levels of circulating MAb enzyme conjugate in the bloodstream due to the long-circulating MAb. The latter, in turn, resulted in excessive enzyme activity in circulation upon administration of the prodrug, which was cleaved by enzyme in the bloodstream, generating high levels of active drug, and high levels of non-specific drug toxicity.
Later, the use of bispecific antibodies (bsAbs) was suggested for application to the ADEPT method. In this approach, a bispecific antibody targeting both a disease-associated antigen with one arm, and an epitope on an enzyme with a second arm would be given to a subject, followed some time later by the enzyme in question, and finally by the prodrug that the enzyme was active against. This comprises a three-step delivery system, absent any clearing agents. Difficulties encountered in the practice of this ADEPT method in the second capture step, that is by the second arm of the bsAb against the enzyme epitope, perhaps due to low affinity of this antibody-antigen complex, led to protocols where the bsAb and the enzyme were mixed together, and administered as a single complex, followed later by the prodrug. This altered approach comprises a two-step delivery system, absent any clearing agents. However, this modified ADEPT method remained fraught with problems preventing its ultimate wide application in patients. These included the utility of the targeting arm of the bsAb, bsAb preparation issues, binding affinity of the second (anti-enzyme epitope) arm of the bsAb, choice of prodrug, efficiency of prodrug cleavage by the enzyme, and, not least, presence of active enzyme in non-target tissues at the time of prodrug administration. The latter leads to unwanted cleavage of prodrug in normal tissues, and, subsequently, untoward toxicity due to the generation of active drug in those tissues. A particular problem was encountered in the cleavage of prodrug to drug in the circulation by active enzyme.
A continuing need therefore exists for methods and compositions that are able to selectively deliver therapy agents to a disease site using an ADEPT approach, without undue dissociation of bsAb and enzyme, and without adversely affecting a therapeutic agent's potency.
The inventors have surprisingly discovered that when a multispecific targeting protein (e.g., a bispecific monoclonal antibody or bispecific antibody fragment) is pre-mixed with a hapten-enzyme covalent conjugate, the resulting complex can be used to localize the enzyme specifically to the site of disease via the targeting arm of the multispecific antibody. The strength of complex binding between the secondary [hapten-binding] arm of the multispecific antibody and the hapten-enzyme conjugate is sufficient to hold the enzyme in a position and concentration suitable for successful ADEPT. The non-covalently bound complex of bsAb/hapten-enzyme remains in circulation for an extended period, showing the stability of the binding between the hapten-binding arm of the bsAb and the hapten-enzyme conjugate. Because the secondary arm of the bsAb is raised against a carefully selected hapten, rather than a non-defined epitope on a particular enzyme, the secondary arm of the bsAb can be carefully screened to have the optimum binding properties. In addition, the same secondary arm-containing bsAb may be used with different enzymes, since the same recognition hapten is being recognized, once the hapten is substituted onto a different enzyme. Such a non-covalently bound complex represents an example of a superior general method for delivery of therapy agents, using ADEPT, to disease tissue targets. This new ADEPT methodology can be adopted to circumvent the aforementioned problems with covalent drug-carrier conjugates, as well as problems seen with earlier versions of the ADEPT concept.
In one aspect, the invention relates to a method for treating target cells, tissues or pathogens in a subject, such as a mammal, comprising administering in sequence:
a therapeutically effective amount of a non-covalently bound complex to said subject thereby forming a target-tissue-localized complex;
wherein said non-covalently bound complex comprises a multispecific targeting protein comprising at least one target-binding site and one hapten-binding site, and a hapten-enzyme covalent conjugate;
wherein said at least one target-binding site is capable of binding to at least one complementary binding moiety on the target cells, tissues or pathogens or on a molecule produced by or associated with said target cells, tissues or pathogens; and
wherein said hapten-binding site is non-covalently bound to the hapten-enzyme covalent conjugate;
optionally, a clearing agent; and
a chemotherapeutic drug or prodrug, capable of being converted to an active drug by the target-tissue-localized complex. More specifically, the chemotherapeutic drug is converted to an active drug by a target-tissue-localized complex that comprises an enzyme.
In another aspect, the invention relates to a kit comprising, in separate containers:
a multispecific targeting protein, comprising at least one target-binding site and a hapten-binding site, pre-mixed with a hapten-enzyme conjugate; and
a chemotherapeutic pro drug.
In yet another aspect, the invention relates to a kit comprising, in separate, suitable containers:
a multispecific targeting protein, comprising at least one target-binding site and a hapten-binding site;
a hapten-enzyme conjugate; and
a chemotherapeutic pro drug;
wherein said multispecific targeting protein, comprising at least one target-binding site and a hapten-binding site and said hapten-enzyme conjugate are mixed immediately prior to use.
In yet another aspect, the invention relates to a method of making a stable non-covalently bound complex that is capable of localizing to a target cell, tissue, or pathogen comprising admixing a multispecific targeting protein comprising at least one target-binding site and a hapten-binding site, and a hapten-enzyme covalent conjugate;
wherein said at least one target-binding site is capable of binding to at least one complementary binding moiety on said target cells, tissues or pathogens or on a molecule produced by or associated with said target cells, tissues or pathogens; and
wherein said hapten-binding site is capable of stably and non-covalently binding said hapten-enzyme conjugate; thereby making a stable non-covalently bound complex.
In still another aspect, the invention relates to a method of treating a subject, comprising administering a therapeutically effective amount of a non-covalently bound complex, said non-covalently bound complex resulting from the pre-mixing of said multispecific targeting protein and a hapten-enzyme conjugate, prior to administration to said subject.
As used herein, the term "subject" refers to any mammal. In one embodiment, the mammal is a human.
Non-Covalently Bound Complex: A Multispecific Targeting Protein and a Hapten-Enzyme Conjugate.
As used herein, the term "targeting protein" is a multispecific binding protein, such as a bispecific antibody, or a recombinantly produced antigen-binding molecule in which two or more of the same or different natural antibody, single-chain antibody or antibody fragment segments with different specificities are linked. The valency of the targeting protein refers to the total number of binding arms or sites the targeting protein has to an antigen or epitope. Thus, depending on the total number of binding arms or sites the targeting protein has to an antigen or epitope, the targeting protein may be monovalent, bivalent, trivalent or multivalent. A multivalent targeting protein has the advantage of multiple interactions in binding to an antigen, thus increasing the avidity of binding to said antigen.
The specificity of the targeting protein refers to how many antigens or epitopes a targeting protein is able to bind. Thus, the targeting protein may be monospecific, bispecific, trispecific or multispecific. A multispecific targeting protein has the advantage of multiple interactions in binding to separate antigens, thus increasing the avidity of binding to the cellular target. Using these definitions, a natural antibody (e.g., an IgG) is bivalent because it has two binding arms but is monospecific because it binds to one antigen.
Monospecific (to target cell), multivalent targeting proteins have more than one binding site for an epitope, but only bind with the same epitope on the same antigen. A second example of a monospecific, multivalent targeting protein is a diabody with two binding sites reactive to the same antigen. The targeting protein may comprise both multivalent and multispecific combinations of different antibody components including multiple copies of the same antibody components.
Examples of multivalent target binding proteins are described in Patent Appl. Ser. No. 60/220,782. Multivalent target binding proteins have been made by cross-linking several Fab-like fragments via chemical linkers. See U.S. Pat. Nos. 5,262,524; 5,091,542 and Landsdorp et al. Euro. J. Immunol. 16: 679-83 (1986). Multivalent target binding proteins also have been made by covalently linking several single chain Fv molecules (scFv) to form a single polypeptide. See U.S. Pat. No. 5,892,020. A multivalent target binding protein which is basically an aggregate of scFv molecules has been disclosed in U.S. Pat. Nos. 6,025,165 and 5,837,242. A trivalent target binding protein comprising three scFv molecules has been described in Krott et al. Protein Engineering 10(4): 423-433 (1997).
In a preferred aspect of the invention, the multivalent and multispecific targeting protein is a bispecific antibody. Such a targeting protein is exemplified by a Fab′�Fab′ fragment, wherein the first Fab′ fragment binds to an anti-tumor cell epitope, and the second Fab′ fragment binds to a low molecular weight hapten. In this embodiment the two distinct specificity Fab′ fragments can be linked through their hinge region thiol groups using commercially available cross-linkers and methods well-known in the art. A second targeting protein is exemplified by a F(ab′)2�Fab′ fragment, wherein the divalent F(ab′)2 fragment binds to an anti-tumor cell epitope, and the monovalent Fab′ fragment binds to a low molecular weight hapten. Similarly, a third targeting protein is exemplified by an intact IgG�Fab′ fragment, wherein the divalent IgG binds to an anti-tumor cell epitope, and the monovalent Fab′ fragment binds to a low molecular weight hapten. Other combinations of specificity and valency of both the anti-target cell arm and the anti-hapten arm may be readily envisaged.
In one preferred aspect of the invention, the multivalent and multispecific (to cellular target and to hapten) targeting protein is a bivalent anti-antigen and monovalent anti-hapten bispecific antibody. Bivalency toward the cellular target better retains the ability of the composition to remain on the cell surface, or associated with the cell for an extended period of time. Monovalency to the hapten limits the amount of cross-linking that can take place with a hapten-enzyme conjugate, and therefore regulates final molecular size. A specific example of such an agent is an anti-CEA�anti-indium-DTPA F(ab′)2�Fab′ bispecific antibody, wherein CEA refers to carcinoembryonic antigen and DTPA refers to diethylenetriaminepentaacetic acid. Further examples will be discussed below.
The target-binding site of a disease-targeting antibody arm is capable of binding to a complementary binding moiety on the target cells, tissues, pathogens or on a molecule produced by, or associated with, the target cell tissue or pathogen. In a preferred aspect of the present invention, the pathogen is selected from the group consisting of a virus, a fungus, a parasite and a bacterium. The complementary binding moieties that are contemplated in one aspect of the present invention include, but are not limited to tumor-associated antigens (TAAs), wherein said antigens are selected from the group consisting of AFP (alpha fetal protein), carbonic anhydrase IX, A3, antigen specific for A33 antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD37, CD45, CD74, CD79a, CD80, HLA-DR, NCA 95, NCA90, HCG and its subunits, CEA (CEACAM-5), CEACAM-6, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, folate receptor, Ba 733, HER2/neu, hypoxia inducible factor (HIF), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, MUC16, PAM- 4-antigen, PSA, PSMA, RS5, 5100, TAG-72, p53, tenascin, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), T101, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, placenta growth factor (P1GF), 17-1A-antigen, an angiogenesis marker, ED-B fibronectin, an oncogene marker, bcl-2, an oncogene product, and other tumor-associated antigens. Recent reports on tumor associated antigens include Mizukami et al., (2005, Nature Med. 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets 5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Ren et al. (2005, Ann. Surg. 242:55-63), each incorporated herein by reference. Specific targeting antibodies of use include, but are not limited to: MN-14 (anti-carcinoembryonic antigen), Mu-9 (anti-colon specific antigen-P), LL2 (anti-CD22), LL1 (anti-CD74), hA20 (anti-CD20) and RS7 (anti-epithelial glycoprotein). Such antibodies encompass chimeric, humanized and human antibodies containing the same CDRs as their corresponding murine antibodies. See U.S. Pat. Nos. 5,874,540; 5,789,554 and 6,187,287. See also pending U.S. patent application Ser. Nos. 10/116,116 (now issued U.S. Pat. No. 7,387,772); 09/337,756 (now U.S. Pat. No. 7,074,405); 60/360,259; and 60/356,132.
The multispecific targeting protein also has an arm referred to as the hapten-binding site or arm. The hapten-binding site is typically an antibody or a hapten-binding antibody fragment and is raised against a defined, low molecular weight hapten. Such low molecular weight haptens include agents such as DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid) and HSG (histamine succinyl glycine moiety):
The antibodies are generally raised after binding of the low molecular weight hapten to an immunogen (e.g., keyhole limpet hemocyanin, or another foreign protein) using methods well know in the art. Specific examples of antibodies that can comprise the hapten-binding site of a multispecific targeting protein include MAbs 734 (anti-diethylenetriaminepentaacetic acid-indium complex; anti-DTPA), 679 (anti-histaminyl succinyl glycyl; anti-HSG) and LG1 (anti-DOTA).
Aside from the MAbs disclosed herein, it can be appreciated that other MAbs can be raised to any hapten or drug by standard methods of making MAbs known to a person skilled in the art. For instance, it is possible to attach a hapten such as HSG to an immunogenic stimulator or adjuvant such a keyhole limpet hemocyanin, and inject the conjugate into immunocompetent animals. Multiple injections are often employed. It must be appreciated that such an approach can lead to several different antibodies with slightly different specificities against the hapten in question, such as HSG. MAbs can recognize different sub-parts of the HSG structure, or different conformations. MAbs may also be obtained that recognize a little more than just the HSG molecule itself, such as recognizing an HSG moiety only when attached to an epsilon amino group of lysine, if indeed, the HSG was initially linked to the KLH (for example) by attachment to an epsilon lysyl amino group on the latter immunogenic protein. Without wishing to be exhaustive, these general procedures and results are well known in the art. It is also then well known art for the isolation of spleen cells producing antibodies from these immunized animals, and their subsequent fusion with myeloma cell lines, to generate hybridomas secreting anti-hapten antibodies. See Kohler G. and Milstein C., Eur. J. Immunol. 6:511-9 (1976); Kohler G. et al., Eur. J. Immunol. 6:292-5 (1976); Harlowe and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; and Kohler G. and Milstein C. Nature 256:495-7 (1975).
Multispecific targeting proteins can be prepared chemically from antibodies that have differing specificity by well-known reactions. Typically, one MAb is activated by reaction with a cross-linking agent, with the latter chosen to react at the first MAb's lysine, reduced cysteine, or oxidized carbohydrate residues. After purification, the activated first MAb is mixed with the second MAb, which then reacts specifically with a second functionality of the original cross-linking agent; most notably via the second MAb's lysine, reduced cysteine or oxidized carbohydrate residues. Multispecific targeting proteins can also be prepared somatically by the quadroma technique. The quadroma technique is a technique wherein a cell line expressing both arms of the bispecific antibody is produced and grown in culture to secrete the bsMAb. Finally, bsMAbs can also be produced conveniently by modern techniques of molecular biology. See, for example Colman, A., Biochem. Soc. Symp. 63: 141-147 (1998); U.S. Pat. No. 5,827,690; and Published U.S. application No. 20020006379, now U.S. Pat. No. 6,962,702.
BsAbs of the types exemplified above can be pre-mixed with several different hapten-enzyme conjugates to produce and deliver an effective therapeutic agent, after appropriate prodrug administration, depending on what the pertinent arm of the bsAb has been raised against. In a preferred embodiment, the enzyme contained in the hapten-enzyme covalent conjugate is selected from the group consisting of an esterase, carboxylesterase, carboxypeptidase, amidase, glucoronidase and galactosidase. Most preferably, the esterase is a carboxylesterase selected from the group consisting of rat, mouse, rabbit, porcine and human carboxylesterase. The enzyme may be produced by recombinant techniques well known in the art (Wolfe, et al. 1999). The enzyme may be produced in yeast, bacteria, plants, insect or animal cells. Preferably, the enzyme has been modified to enhance its catalytic properties (Wolfe et al, 1999). The modification may be performed via site-directed mutagenesis. See U.S. Pat. Nos. 5,352,594 and 5,912,161 for a general discussion of site-directed mutagenesis. In any case, the desired effect of the mutagenesis is to reduce the Michaelis constant of the enzyme, enabling more efficient enzyme activity at lower concentrations of prodrug substrate. It is preferred that the multispecific targeting protein binds to both its antigenic target and to its hapten target via the target binding site and the hapten-binding site, respectively, with a dissociation constant of about 10−7; more preferably about 10−9.
Haptens can be attached to enzymes in several ways. For instance, the DTPA hapten can be coupled to the enzyme carboxylesterase at certain individual positions on the enzyme to give the hapten-enzyme covalent conjugate. Most simply the commercially available precursor DTPA dianhydride is added to a solution of enzyme in an appropriate buffer, at pH 7-9. After a reaction of from 1-16 hours, using an appropriate molar excess of DTPA-dianhydride, one or more units of DTPA are attached to the enzyme, by reaction of the latter's lysyl residues with one anhydride group of the precursor. The DTPA-enzyme conjugate is separated from unreacted, hydrolyzed DTPA and buffer components by standard methods for effecting such separations, such as ammonium sulfate precipitation, diafiltration or size-exclusion or ion-exchange chromatography. To obtain the bsAb-hapten-enzyme conjugate the hapten-enzyme covalent conjugate is then mixed with a bsAb, such as MN-14�734 bsAb (anti-CEA�anti-DTPA) to give a non-covalently bound complex wherein the target-binding site capable of binding to a complementary binding moiety on the target cells is MN-14. A typical complex might then be: MN-14�734 bsAb/DTPA-carboxylesterase. The bsAb and the hapten-enzyme conjugate may be mixed together in ratios of from 5:1 to 1:5, or more preferably in ratios of from 2:1 to 1:2. However, these ratios are not limiting and other ratios, for example 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5, or any range in between any of these ratios, may be utilized. The complex may be made immediately prior to use, or it may be made in advance and stored under appropriate conditions until required. It may also be frozen for shipping and future use, or formulated for long-term storage by lyophilization. Such methods are well known in the art.
The hapten-enzyme conjugate may also be made using an alternate approach, designed to attach two hapten recognition units to the enzyme in one chemical reaction. In this approach, an intermediate comprising two such hapten recognition units is attached to a short peptide carrier backbone that also incorporates a group for activation and coupling to the enzyme. The agent has the general formula: X-peptide(-X)-(reactive group); where the peptide is 2-10 amino acid residues in length, preferably 2-5 amino acid residues in length, most preferably, the peptide is 3-4 amino acid residues in length. The X moieties are recognition hapten residues mentioned previously, exemplified by In-DTPA, DOTA or HSG sub-units; and the reactive moiety comprises a functionality that can be coupled to the enzyme without interference from the rest of the bivalent recognition conjugate. An Example of such a structure is Ac-NH-Lys(HSG)-Tyr-Lys(HSG)-COOH; a tripeptide of two lysyl residues and one tyrosyl residue, linked together by amide bonds, and blocked on its alpha amino group by an unreactive group such as an acetyl residue. The amino acids may be in the L- or the D-conformation. Each lysyl residue, though its epsilon amino group, is attached to an HSG recognition unit. The reactive moiety in this instance is a carboxyl group that can be further activated via an anhydride, active ester or other such activating agent, for coupling to free amino groups on an enzyme.
A second similar example of such a structure is 4(4-N-maleimidomethyl)cyclohexanecarboxyamido-Lys(DTPA)-Tyr-Lys(DTPA)-CONH2; a tripeptide of two lysyl residues and one tyrosyl residue, linked together by amide bonds, and blocked on its carboxyl terminal group by an unreactive group such as an amide residue. The amino acids may be in the L- or the D-conformation. Each lysyl residue is attached to a DTPA recognition unit. The reactive moiety in this instance is a maleimido group that might be coupled to free thiol groups on an enzyme, wherein the free thiol groups are present endogenously, or are placed there by prior reaction of the enzyme with a thiolating agent such as Traut's reagent.
Many more such compositions can be envisaged as useful within the context of the current invention. See for example published U.S. application No. 20020006379, now U.S. Pat. No. 6,962,702, and pending U.S. application Ser. No. 09/337,756, now U.S. Pat. No. 7,074,405, each incorporated herein by reference in their entirety.
After administration, localization to the site of disease, and substantial clearance from normal tissues of the bsAb/hapten-enzyme complex, a drug or prodrug substrate to the enzyme in question may be given. For example, with a CEA-expressing tumor, the above MN-14�734 bsAb, pre-complexed with DTPA-carboxylesterase is given, allowed to localize to CEA-expressing tumor sites, and clear normal tissues, before the prodrug CPT-11 (irinotecan) (a substrate for carboxylesterase) is given. The non-covalently bound bsAb-hapten-enzyme complex that has localized at the tumor, activates the subsequently administered prodrug specifically at the site of the tumor. A variety of chemotherapeutic agents or prodrugs of chemotherapeutic agents may be used in the practice of the preferred embodiments of the present invention for treatment of subjects. Such chemotherapeutic agents include, but are not limited to, adriamycin, actinomycin, calicheamycin, epothilones, maytansine, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol and other taxanes, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin(s), neomycin, and podophyllotoxin(s). Anti-metabolites such as cytosine arabinoside, amethopterin; anthracyclines; vinca alkaloids and other alkaloids; antibiotics, demecolcine; etopside; mithramycin; and other anti-tumor alkylating agents are also contemplated for use in the present invention.
Preferred prodrugs of the preferred embodiments are those derived from the drugs selected from the group consisting of camptothecin, doxorubicin, taxol, actinomycin, maytansine, calicheamycin and epothilones.
The term "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. The prodrug, for instance, may be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. See Harper, "Drug Latentiation" in Jucker, ed. Progress in Drug Research 4:221-294 (1962); Morozowich et al., "Application of Physical Organic Principles to Prodrug Design" in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APHA Acad. Pharm. Sci. (1977); Bioreversible Carriers in Drug in Drug Design, Theory and Application, E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier (1985); Wang et al. "Prodrug approaches to the improved delivery of peptide drug" in Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998) "The Use of Esters as Prodrugs for Oral Delivery of (3-Lactam antibiotics," Pharm. Biotech. 11:345-365; Gaignault et al. (1996) "Designing Prodrugs and Bioprecursors I. Carrier Prodrugs," Pract. Med. Chem. 671-696; Asgharnejad, "Improving Oral Drug Transport", in Transport Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218 (2000); Balant et al., "Prodrugs for the improvement of drug absorption via different routes of administration", Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko, "Involvement of multiple transporters in the oral absorption of nucleoside analogues", Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne, "Fosphenyloin (Cerebyx)", Clin. Neuropharmacol. 20(1): 1-12 (1997); Bundgaard, "Bioreversible derivatization of drugs—principle and applicability to improve the therapeutic effects of drugs", Arch. Pharm. Chemi 86(1): 1-39 (1979); Bundgaard H. "Improved drug delivery by the prodrug approach", Controlled Drug Delivery 17: 179-96 (1987); Bundgaard H. "Prodrugs as a means to improve the delivery of peptide drugs", Adv. Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al. "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Adv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. "Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting", Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81, (1985); Farquhar D, et al., "Biologically Reversible Phosphate-Protective Groups", J. Pharm. Sci., 72(3): 324-325 (1983); Freeman S, et al., "Bioreversible Protection for the Phospho Group: Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl) Methylphosphonate with Carboxyesterase," J. Chem. Soc., Chem. Commun., 875-877 (1991); Friis and Bundgaard, "Prodrugs of phosphates and phosphonates: Novel lipophilic alpha-acyloxyalkyl ester derivatives of phosphate- or phosphonate containing drugs masking the negative charges of these groups", Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangway et al., "Pro-drug, molecular structure and percutaneous delivery", Des. Biopharm. Prop. Prodrugs Analogs, [Symp. J Meeting Date 1976, 409-21. (1977); Nathwani and Wood, "Penicillins: a current review of their clinical pharmacology and therapeutic use", Drugs 45(6): 866-94 (1993); Sinhababu and Thakker, "Prodrugs of anticancer agents", Adv. Drug Delivery Rev. 19(2): 241-273 (1996); Stella et al., "Prodrugs. Do they have advantages in clinical practice?", Drugs 29(5): 455-73 (1985); Tan et al. "Development and optimization of anti-HIV nucleoside analogs and prodrugs: A review of their cellular pharmacology, structure-activity relationships and pharmacokinetics", Adv. Drug Delivery Rev. 39(1-3): 117-151 (1999); Taylor, "Improved passive oral drug delivery via prodrugs", Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino and Borchardt, "Prodrug strategies to enhance the intestinal absorption of peptides", Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus, "Concepts for the design of anti-HW nucleoside prodrugs for treating cephalic HIV infection", Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999); Waller et al., "Prodrugs", Br. J. Clin. Pharmac. 28: 497-507 (1989).
A clearing agent may be optionally added after administration of the noncovalently bound multispecific antibody-hapten-enzyme complex to a subject. The clearing agent is preferably an antibody directed against an epitope of the multispecific targeting protein/hapten-enzyme complex. Most preferably, the clearing agent is an anti-idiotic antibody, a carbohydrate-derivatized anti-idiotic antibody or a galactosylated anti-idiotypic antibody to the multispecific targeting protein.
Non-proteinaceous polymers can also serve as the backbone onto which other drugs or prodrugs useful in the present invention may be attached. The polymeric material serves to detoxify and solubilize the active drug. For instance a co-polymer consisting of (Lys)m (Glu)n(Taxol)y (wherein m is an integer from 10-500, n is an integer from 10-500, and y is an integer from 1-50 (SEQ ID NO: 1)) can be applied in this manner, being given after the injection, localization and clearance of the multispecific antibody-hapten-enzyme complex. In this instance, the enzyme in question would comprise an esterase, capable of cleaving the ester bond between taxol and the gamma-carboxyl groups of the multi-glutamic acid units. This type of prodrug is based on the utility of polymeric material to carry active drugs in circulation for an extended period of time. See Auzenne et al., Clin Cancer Res. 8: 573 (2002) and Li et al., Cancer Res., 1998. Other drugs, such as camptothecins may be used in a similar manner, and other polymers such as poly-N-(2-hydroxypropyl)methacrylamide (HPMA) may also be applied as carriers. The invention also contemplates the incorporation of unnatural amino acids, e.g., D-amino acids, into the non-proteinaceous polymers. The invention further contemplates other backbone structures such as those constructed from non-natural amino acids. See for example, published U.S. application No. 20030026764, now abandoned.
In another aspect, the invention relates to a method of making a stable target-tissue-localized complex comprising pre-mixing a multispecific targeting protein comprising at least one target-binding site and a hapten-binding site, and a hapten-enzyme covalent conjugate;
wherein said at least one target-binding site is capable of binding to at least one complementary binding moiety on the target cells, tissues or pathogens or on a molecule produced by or associated with said target cells, tissues or pathogens; and
wherein said hapten-binding site is capable of stably and non-covalently binding a hapten-enzyme conjugate; thereby forming a stable target-tissue-localized complex.
Formulations and Kits
The multispecific targeting protein and hapten-enzyme covalent conjugate that comprises the non-covalently bound complex preferably also comprise a pharmaceutically acceptable carrier or excipient. A pharmaceutically acceptable carrier is a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An excipient is an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol derivatives.
One aspect of the present invention relates to a kit comprising, in suitable containers, separate or together:
a multispecific targeting protein, comprising a target tissue-binding site and an hapten-binding site, pre-mixed with a hapten-enzyme conjugate; and
a chemotherapeutic pro drug.
Another aspect of the invention relates to a kit comprising, in separate, suitable containers:
a multispecific targeting protein, comprising at least one target-binding site and a hapten-binding site;
a hapten-enzyme conjugate; and
a chemotherapeutic pro drug;
wherein said multispecific targeting protein, comprising at least one target-binding site and a hapten-binding site and said hapten-enzyme conjugate are mixed immediately prior to use.
The kit, may comprise the non-covalently bound complex and a pharmaceutically acceptable carrier or excipient. Likewise, the kit may comprise the prodrug in a pharmaceutically acceptable carrier or excipient. In a preferred embodiment of the present invention, the kit comprises a bispecific antibody such as anti-CEA�anti-indium-DTPA-F(ab′)2. The bispecific antibody is mixed with an equimolar amount of the enzyme-hapten conjugate DTPA-carboxylesterase. The kit can contain from about 1-10,000 mg of the mixture. The kit can be stored as a sterile solution, frozen at −20 to −80� C., or it can be lyophilized to powder form for long-term storage. In one embodiment, these formulations could include a preformed single vial kit comprising multispecific antibody-hapten-enzyme conjugate, or two separate vials containing multispecific antibody, and hapten-enzyme, respectively, which are then mixed prior to administration. From a formulation and stability perspective, the hapten-enzyme may be kept separate for long-term storage, and these determinations need to be made empirically, for each individual application of the technology.
An amount of the non-covalently bound complex necessary for treating target cells, tissues or pathogens in a subject when provided to a subject is a "therapeutically effective" amount. In order to treat the target cells, tissues or pathogens, it is desirable to provide from about 0.001 to about 10,000 micromoles of non-covalently bound complex per kilogram of subject weight. This dosage may be administered over a period from about 1 minute to about 4 hours, by any suitable means, but prior to the administration of the chemotherapeutic drug or prodrug. The non-covalently bound complex of the present invention may be dissolved in any physiologically tolerated liquid in order to prepare an administrable amount. It is preferable to prepare such a solution of the non-covalently bound complex by dissolving the non-covalently bound complex in normal saline, phosphate buffered saline (pH from about 5 to about 8), acetate buffered saline (pH from about 4 to about 7), phosphate buffer (pH from about 5 to about 8), or acetate buffer (pH from about 4 to about 7). Buffered concentrations in the 0.02 to 2 molar range are acceptable.
An amount of the chemotherapeutic drug or prodrug necessary to treat target cells, tissues or pathogens in a subject when provided after the administration of the non-covalently bound complex to a subject is a "therapeutically effective" amount. In order to treat the target cells, tissues or pathogens, it is desirable to provide from about 0.001 to about 10,000 μmol of non-covalently bound complex per kilogram of subject weight. This dosage may be administered over a period from about 1 minute to about 4 hours, by any suitable means, but following the administration of the non-covalently bound complex. The chemotherapeutic drug or prodrug of the preferred aspects of the present invention may be dissolved in any physiologically tolerated liquid in order to prepare an administrable amount. It is preferable to prepare such a solution of the non-covalently bound complex by dissolving the non-covalently bound complex in normal saline, phosphate buffered saline (pH from about 5 to about 8), acetate buffered saline (pH from about 4 to about 7), phosphate buffer (pH from about 5 to about 8), or acetate buffer (pH from about 4 to about 7). Buffered concentrations in the 0.02 to 2 molar range are acceptable. Drugs or prodrugs may be administered in the ways that they are usually administered when given as independent active entities. For instance, hydrophobic drugs or prodrugs may be given in dextrose solutions or as admixtures with cremophor.
Suitable routes of administration of the non-covalently bound complex and the chemotherapeutic drug or prodrug include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections. The preferred routes of administration are parenteral. Alternatively, one may administer the bsAb/enzyme-hapten complex and the drug or prodrug in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor.
The ordinary skilled artisan will appreciate that the pre-mixing of the multispecific targeting protein and the hapten-enzyme conjugate, prior to administration to a subject, can be done with immediately before administration, or, it can be done well in advance.
In another aspect, the invention relates to a method of treating a subject, comprising administering a therapeutically effective amount of a non-covalently bound complex, said non-covalently bound complex resulting from the pre-mixing of said multispecific targeting protein and a hapten-enzyme conjugate, prior to administration to a subject.
Diseases that may be treated using the pre-mixed multispecific targeting proteins and hapten-enzyme conjugates of the preferred embodiments of the present invention include, but are not limited to malignancies. These may include all solid and non-solid tumor cancers. In the case of the latter, B-cell cancers, or T-cell cancers can be treated (e.g., non-Hodgkins lymphoma, T-cell lymphoma or chronic lymphocytic leukemia). Equally, solid tumors may be treated using the current compositions and methods. These include, but are not limited to, adenocarcinomas and sarcomas. Major cancers of endodermally-derived digestive system epithelia, and cancers of the breast, prostate and lung are contemplated and treatable using this approach. In preferred applications diseases expressing antigens such as AFP (alpha fetal protein), HCG (human chorionic gonadotropin), EGP-1, EGP-2, CD37, CD74, colon-specific antigen-p (CSAp), carcinoembryonic antigen (CEA), CD19, CD20, CD21, CD22, CD23, CD30, CD74, CD80, HLA-DR, Ia, MUC 1, MUC 2, MUC 3, MUC 4, EGFR, HER 2/neu, PAM-4, TAG-72, EGP-1, EGP-2, A3, KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGF, necrosis antigens, IL-2, T101 and MAGE can be targeted with an appropriate antigen-targeting antibody arm on the multispecific antibody. Specific targeting antibodies include, but are not limited to: MN-14 (anti-carcinoembryonic antigen), Mu-9 (anti-colon specific antigen-P), LL2 (anti-CD22), LL1 (anti-CD74), hA20 (anti-CD20) RS7 (anti-epithelial glycoprotein-1). Such antibodies encompass chimeric, humanized and human antibodies containing the same CDRs as their corresponding murine antibodies.
Other diseases than cancer can also be targeted using these multispecific antibody/hapten-enzyme conjugates. For example, anti-CD19, anti-CD20, anti-CD22 and anti-CD74 antibodies can be used to treat immune dysregulation diseases and related autoimmune diseases, including Class III autoimmune diseases such as immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sjogren's syndrome, multiple sclerosis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, (repeat), primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.
The following examples are meant to be illustrative of the methods, compositions and uses of the invention, and are not intended to be limitative.
Preparation of a Carboxylesterase-DTPA Conjugate
Two vials of rabbit liver carboxylesterase (about 8.5 mg protein content/vial) are reconstituted with 2.3 mL of 50 mM potassium phosphate buffer pH 7.5, and the solution is made 4.2 mM in DTPA using 0.1 mL of a 0.1 M stock solution of DTPA pH 6.7. The pH of the resultant solution is adjusted to be in the 7.7-7.8 range, and then reacted with 10 mg of cyclic DTPA dianhydride. After 1 h of stirring at the room temperature, the reaction mixture is passed through two successive SEC columns equilibrated in 0.1 M sodium phosphate pH 7.3. The eluate is further purified by preparative HPLC on a TSK G3000SW column using 0.2 M sodium phosphate pH 6.8, at 4 mL/min flow, as the eluent. The purified conjugate is made 0.1 M in sodium phosphate pH 6.8, and concentrated. The DTPA-to-carboxylesterase molar substitution ratio, determined by a metal-binding assay, is estimated to be in the range of 2.95-to-1 to 4.43-to-1.
Preparation of hMN-14 IgG�734 Fab′ Bispecific Antibody
HMN-14 IgG (8.45 mg, MW 150K) was derivatized with a 1.8-fold molar excess of sulfo-SMCC, at pH 7.21, for 45 minutes at ambient temperature. The product was purified by centrifuged SE column ('spin column') of Sephadex G50/80 in 0.1 M sodium phosphate, pH 6.5. The maleimide content was determined to be 0.93 moles per mole of IgG, by reacting with a known excess of 2-mercaptoethanol, followed by the determination of unused thiol by Ellman's assay. Separately, 734 F(ab′)2 was reduced with 0.1 M cysteine (˜100-fold molar excess of cysteine) in 20 mM Hepes buffer-150 mM sodium chloride-10 mM EDTA, pH 7.3. The reduction was carried out for 50 min at 37� C. (bath) under argon flush. The reduced material was purified by two successive spin columns on Sephadex G50/80 in 0.1 M sodium phosphate-S mM EDTA, pH 6.5. The hMN-14-maleimide was reacted with 2-fold excess of 734 Fab′, and incubated at ambient temperature for 1 h. The conjugate was then reacted with a 40-fold molar excess of N-ethylmaleimide for 40 min. The material was subjected to preliminary purification on spin column of Sephadex G50/80 in 0.1 M sodium phosphate, pH 7.3. The eluate from this purification was applied to a column of 3 mL of DTPA-Affigel, which was sequentially eluted with 0.2 M sodium phosphate pH 6.8 and 0.1 M EDTA, pH 3.8. Pooled EDTA fractions were dialyzed against 0.2 M sodium phosphate pH 6.8, with 2 buffer changes. The sample was applied to a preparative SE HPLC column (TSK G3000SW), with 0.2 M sodium phosphate pH 6.8 at 4 mL/min as running buffer. The major product was separated, and was found pure by SE HPLC (ret. time 9.58 min on analytical SE HPLC, 0.2 M sodium phosphate at 1 mL/min flow). Recovery: 7.69 mg. MW by MALDI mass spectral analysis was 196, 803.
Biodistribution of a Pre-Mixed Complex Comprising an Indium-Labeled Carboxylesterase-DTPA Conjugate and the bsMAb hMN-14�734 (IgG�Fab′)
This example demonstrates the biodistribution of a pre-mixed complex of hMN-14�734 Fab′ (hMN-14 is a humanized antibody of MN-14 (carcinoembryonic antigen; anti-CEA), and an indium-DTPA-carboxylesterase conjugate. Carboxylesterase-DTPA is radiolabeled for tagging purposes with indium-111 radionuclide, using commercially available In-111 acetate. Briefly, In-111 chloride was buffered with 3-times the volume of 0.5 M sodium acetate, pH 6.1; 0.12 mg of CE-DTPA was mixed with 0.25 mCi of In-111 acetate, and incubated for 40 minutes. Then added −0.01 mL of cold indium acetate [In acetate: prepared from 0.005 mL of 1.97�10−2 M indium chloride, 0.045 mL of water and 0.15 mL of 0.5 M sodium acetate, pH 6.1.] After 20 minutes, the solution was made 10 mM in EDTA, incubated for 10 min. ITLC analyses showed 98% of radioactivity associated with carboxylesterase. The pre-mixed hMN-14�734 Fab′/In-1′-In-DTPA-carboxylesterase complex is administered to hamsters and nude mice bearing GW-39 human tumor xenografts.
Tables 1-6 show that the binding between of the carboxylesterase-DTPA conjugate and the corresponding bispecific antibody is stable in vivo, and that the In-111/In-DTPA-carboxylesterase conjugate can be effectively localized and retained at the tumor sites by its complexation with the hMN-14�734 (IgG�Fab′) bsAb.
ADEPT Therapy Using a Pre-Mixed Complex Comprising an Indium (In)-DTPA Carboxylesterase conjugate and the bsMAb hMN-14�734 (IgG�Fab′)
Male hamsters (body weight: ˜75 g) are given GW-39 human tumor xenografts by injection of a 20% v/v GW-39 tumor cell suspension intramuscularly on the animals' right thigh. After 3 days, a 2:1 premixed complex of mMN-14 F(ab)2�m734Fab′ and Indium-DTPA-carboxylesterase, at a dose of 0.75 mg of bsAb, corresponding to 200 enzyme units per kg body weight, is administered. Five days post-injection of bsAb/In-DTPA-carboxylesterase, a maximum tolerated dose (8 mg/75 g body weight; determined earlier) of the pro drug, CPT-11, is given. A positive control group is given CPT-11 alone and an untreated group are also included in the study. Tumor growth in untreated animals is out of control at 3-4 weeks post-implantation of tumor cells, and animals are sacrificed for humane reasons. Mean tumor volumes are similar for the bsAb/In-DTPA-carboxylesterase and the positive control (CPT-11 alone) at 5 weeks, and out to 9 weeks post-implantation of tumor cells. However, the bsAb/In-DTPA-carboxylesterase treated group continues to show growth inhibition over the next five weeks, while the mean tumor volumes for the group given CPT-11 alone increase during the same period. The relative mean tumor volume for the bsAb/In-DTPA-carboxylesterase treated group at week 14 is similar to the mean tumor volume at week 9 for the positive control, CPT-11-alone-treated animals. This demonstrates a 5-week advantage in tumor growth control when applying an ADEPT approach using bsAb/In-DTPA-carboxylesterase pretargeting.
Preparation of Carboxylesterase-IMP222 ("CE-IMP222')
IMP222 is a di-DTPA-containing peptide with the cysteine thiol available for conjugation to maleimide-appended carboxylesterase. IMP222: Ac-Cys-Lys(DTPA)-Tyr-Lys (DTPA)-NH2 (SEQ ID NO: 2). Carboxylesterase (0.0245 umol) was derivatized with a 17.5-fold molar excess of sulfo-SMCC [sulfosuccinimidyl 4(N-maleimidomethyl)-1-cycclohexane carboxylate] in 0.1 M sodium phosphate, pH 7.3, at the ambient temperature for 45 minutes. The product was purified on a 2-mL centrifuged SE column ('spin column') of Sephadex G50/80 in 0.1 M sodium phosphate, pH 7.3. The solution of the product was made 1 mM in
EDTA, and reacted with a 20-fold molar excess of IMP-222 contained in 0.1 M sodium phosphate-5 mM EDTA, pH 6.5, for 45 minutes at the ambient temperature. The product was purified by 'spin column' of Sephadex G50/80 in 0.1 M sodium phosphate, pH 7.3. Metal binding analysis using excess of indium acetate spiked with radioactive indium, gave an average of 4.5 DTPAs/conjugate in two determinations, or average of 2.25 IMP222 moieties per conjugate. Test labeling with In-111 acetate gave 94% incorporation as assayed by ITLC. The material was completely complexed by mixing with a 5-fold molar excess of F6�734 Fab′ Fab′ bispecific antibody, as judged by the shift of the HPLC peak to the higher MW region of the complex.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usage and conditions without undue experimentation. All patents, patent applications and publications cited herein are incorporated by reference in their entirety.
European Patent No. 0595743.
Auzenne, E., Donato, N. J., Li, C., Leroux, E., Price, R. E., Farquhar, D. and Klostergaard, J. Superior therapeutic profile of poly-L-glutamic acid-paclitaxel copolymer compared with taxol in xenogeneic compartmental models of human ovarian carcinoma. Clin. Cancer Res., 8:573-581, 2002.
Bagshawe, K. D. Antibody-directed enzyme prodrug therapy for cancer: its theoretical basis and application. Mol. Med. Today, 1:424-431, 1995.
Bagshawe, K. D., Sharma, S. K., Springer, C. J., Antoniw, P., Boden, J. A., Rogers, G. T., Burke, P. J., Melton, R. G. and Sherwood, R. F. Antibody directed enzyme prodrug therapy (ADEPT): clinical report. Dis. Markers, 9:233-238, 1991.
Barbet et al., Pretargeting with the affinity enhancement system for radioimmunotherapy. Cancer Biother. Radiopharm., 14:153-166, 1999.
Biela, B. H., Khawli, L. A., Hu, P. and Epstein, A. L. Chimeric TNT-3/human betaglucuronidase fusion proteins for antibody-directed enzyme prodrug therapy (ADEPT). Cancer Biother, Radiopharm., 18:339-353, 2003.
Bonardi et al., Delivery of saporin to human B-cell lymphoma using bispecific antibody: Targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing. Cancer Res., 53:3015-3021, 1993.
Casey et al., Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab′ fragments. Br. J. Cancer., 74:1397-1405, 1996.
Chen, B. M., Cheng, T. L., Tzou, S. C. and Roffler, S. R., "Potentiation of antitumor immunity by antibody-directed enzyme prodrug therapy," Int. J. Cancer, 94:850-858, 2001.
Darks, M. K., Morton, C. L., Krull, E. J., Cheshire, P. J., Richmond, L. B., Naeve, C. W., Pawlik, C. A., Houghton, P. J. and Potter, P. M. Comparison of activation of CPT-11 by rabbit and human carboxylesterases for use in enzyme/prodrug therapy. Clin. Cancer Res., 5:917-924, 1999.
Deckert, P. M., Renner, C., Cohen, L. S., Jungbluth, A., Ritter, G., Bertino, J. R., Old, L. J. and Welt, S. A33scFv-cytosine deaminase: a recombinant protein construct for antibody-directed enzyme-prodrug therapy. Br. J. Cancer, 88:937-939, 2003.
de Bont, D. B., Leenders, R. G., Haisma, H. J., van der Meulen-Muileman, I. and Scheeren, H. W. Synthesis and biological activity of beta-glucuronyl carbamate-based prodrugs of paclitaxel as potential candidates for ADEPT. Bioorg. Med. Chem., 5:405-414, 1997.
de Graaf, M., Boven, E., Scheeren, H. W., Haisma, H. J. and Pinedo, H. M. Betaglucuronidase-mediated drug release. Curr. Pharm. Des., 8:1391-1403, 2002.
Denny, W. A. Prodrug strategies in cancer therapy. Eur. J. Med. Chem., 36:577-595, 2001.
Francis, R. J., Sharma, S. K., Springer, C., Green, A. J., Hope-Stone, L. D., Sena, L., Martin, J., Adamson, K. L., Robbins, A., Gumbrell, L., O'Malley, D., Tsiompanou, E., Shahbakhti, H., Webley, S., Hochhauser, D., Hilson, A. J., Blakey, D., Begent, R. H. A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours. Br. J. Cancer, 87:600-607, 2002.
French. Production of bispecific and trispecific F(ab)2 and F(ab)3 antibody derivatives. Methods Mol. Biol., 80:121-134, 1998.
Gestin et al., Two-step targeting of xenografted colon carcinoma using a bispecific antibody and 188Re-labeled bivalent hapten: biodistribution and dosimetry studies. J. Nucl. Med., 42:146-153, 2001.
Hillairet de Boisferon et al., Enhanced targeting specificity to tumor cells by simultaneous recognition of two antigens. Bioconjug Chem., 11:452-460, 2000.
Houba, P. H., Boven, E., van der Meulen-Muileman, I. H., Leenders, R. G., Scheeren, J. W., Pinedo, H. M. and Haisma, H. J. Pronounced antitumor efficacy of doxorubicin when given as the prodrug DOX-GA3 in combination with a monoclonal antibody beta-glucuronidase conjugate. Int. J. Cancer, 91:550-554, 2001.
Jung M. Antibody directed enzyme prodrug therapy (ADEPT) and related approaches for anticancer therapy. Mini Rev. Med. Chem., 1:399-407, 2001.
Kan et al., Thioether-bonded constructs of Fab′gamma and Fc gamma modules utilizing differential reduction of interchain disulfide bonds. J Immunol., 166:1320-1326, 2001.
LeDoussal et al., Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen positive mouse B cells with monoclonal antibody conjugate cocktails. J. Immunol., 146:169-175, 1991.
Li, C., Yu, D. F., Newman, R. A., Cabral, F., Stephens, L. C., Hunter, N., et al. Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate. Cancer Res., 58:2404-2409, 1998.
Melton, R. G., Boyle, J. M., Rogers, G. T., Burke, P., Bagshawe, K. D. and Sherwood, R. F. Optimisation of small-scale coupling of A5B7 monoclonal antibody to carboxypeptidase G2. J. Immunol Methods, 158:49-56, 1993.
Schott et al., Preparation, characterization, and in vivo biodistribution properties of synthetically cross-linked multivalent antitumor antibody fragments. Bioconjug. Chem., 4:153-165, 1993.
Schoonjans et al., Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives.
J. Immunol., 165:7050-7057, 2000.
Schoonjans et al., A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain. Biomol. Eng., 17:193-202, 2001.
Sharma, S. K., Bagshawe, K. D., Springer, C. J., Burke, P. J., Rogers, G. T., Boden, J. A., Antoniw, P., Melton, R. G. and Sherwood R F. Antibody directed enzyme prodrug therapy (ADEPT): a three phase system. Dis. Markers, 9:225-231, 1991.
Sharma, S. K., Bagshawe, K. D., Burke, P. J., Boden, R. W. and Rogers, G. T. Inactivation and clearance of an anti-CEA carboxypeptidase G2 conjugate in blood after localisation in a xenograft model. Br. J. Cancer, 61:659-662, 1990.
Spooner, R. A., Friedlos. F., Maycroft, K., Stribbling, S. M., Roussel, J., Brueggen, J., Stolz, B., O'Reilly, T., Wood, J., Matter, A., Marais, R. and Springer, C. J. A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs. Br. J. Cancer, 88:1622-1630, 2003.
Springer, C. J., Antoniw, P., Bagshawe, K. D. and Wilman, D. E. Comparison of half-lives and cytotoxicity of N-chloroethyl-4-amino and N-mesyloxyethyl-benzoyl compounds, products of prodrugs in antibody-directed enzyme prodrug therapy (ADEPT). Anticancer Drug Des., 6:467-479, 1991.
Springer, C. J. and Niculescu-Duvaz, I. Antibody-directed enzyme prodrug therapy (ADEPT): a review. Adv. Drug Deliv. Rev., 26:151-172, 1997.
Tietze, L. F., Herzig, T., Fecher, A., Haunert, F. and Schuberth, I. Highly selective glycosylated prodrugs of cytostatic CC-1065 analogues for antibody-directed enzyme tumor therapy. Chembiochem. 2:758-765, 2001.
Tietze, L. F., Lieb, M., Herzig, T., Haunert, F. and Schuberth, I. A strategy for tumor-selective chemotherapy by enzymatic liberation of seco-duocarmycin SA-derivatives from nontoxic prodrugs. Bioorg. Med. Chem., 9:1929-1939, 2001.
Werlen et al., Preparation of a trivalent antigen-binding construct using polyoxime chemistry: improved biodistribution and potential for therapeutic application. Cancer Res., 56:809-815, 1996.
Wolfe, L. A., Mullin, R. J., Laethem, R., Blumenkopf, T. A., Cory, M., Miller, J. F., Keith, B. R., Humphreys, J. and Smith, G. K. Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodmgs of methotrexate and the thymidylate synthase inhibitors GW 1031 and GW1843. Bioconjug. Chem., 10:38-48, 1999.
Xu, G. and McLeod, H. L. Strategies for enzyme/prodrug cancer therapy. Clin. Cancer Res., 7:3314-3324, 2001.
Patent Citations
Non-Patent Citations
Legal Events
Original Image


text:
The "Not Guilty" verdict was read close to 10pm on Saturday night. Immediately, social media became the outlet to express emotion about the unfair ruling and even though this case is as simple to some as black and white, a lot of White people were equally outraged.
Popular actors, musicians, hosts and other influential people didn't shy away from the race factor or how heart-wrenching the decision and tragedy is.
MUST READ: Wait, What?! George Zimmerman's Brother Says, 'Trayvon Martin Was Looking To Procure Firearms' [VIDEO]
MUST READ: President Obama Responds To George Zimmerman's Not-Guilty Verdict
Like HelloBeautiful on Facebook to stay on top of your favorite black celebrities:
Latest Galleries
- Meet Janet Jackson's Dancers For The Unbreakable Tour!
- Hello Handsome: Intellectual BAEs Who Mentally Seduce Us
- Happy Birthday Royalty! The Most Adorable Photos Of Chris Brown's Baby Girl!
- Break Ups To Make Ups: The Best Of The Worst Celebrity Love Affairs
- BET Awards 2014
- Billboard Music Awards
- North West Is The Cutest, Most Stylish Baby Of All Time!


text:
Everybody Wants a Bailout
Monday, July 6, 2009
Matt Chaban.
With the news today, reported by The Observer, that Larry Silverstein has begun legal proceedings against the Port Authority to end the gridlock at Ground Zero, as well as the developments two weeks prior at Atlantic Yards, it seems obvious to us what's going on here. Having witnessed the financial titans across town receive hundreds of billions of dollars in bailout money, these developers now want theirs. Granted, so did Larry Flint and the porn industry, but the comparison bears consideration. Read More
Shovels in the Ground
Tuesday, April 14, 2009
Matt Chaban.
Yesterday, President Obama made a visit to the Department of Transportation to applaud them and the rest of the nation for their work spending those stimulus dollars, marking the occasion of the 2,000th infrastructure project to be approved for Federal stimulus money. In his speech, the president joked that something unusual had happened at DOT and throughout the land: "We can utter a sentence rarely heard in recent years: This government effort is coming in ahead of schedule and under budget." Read More
Stimulus, Coming to a Street Near You
Tuesday, March 31, 2009
Matt Chaban.
First Recovery.gov, now the NYC Stimulus Tracker. Yesterday, when Mayor Michael Bloomberg unveiled the $1.1 billion in new infrastructure spending resulting from the city's cut of the federal stimulus bill, he also announced the creation of a special website to lend transparency to the process, not unlike the model set out by our dear mayor. (Judging by WNYC and ProPublica's Shovelwatch map, though, everyone's getting in on the act, with all but five states and numerous municipalities launching such sites.) Read More
Detroit Pick Me Up
Tuesday, March 3, 2009
Alan G. Brake.
The collapse of Detroit has been a subject of fascination for artists, architects, and urbanists. In the current economic environment, the symbolism of the city's decline threatens to overshadow the human and physical realities of the place. And while few would argue that the city will ever return to its height, it is nice to be able to show a project that disrupts the city-in-free-fall narrative, such as the new Mercury Coffee Bar, designed by Andrew Zago. No architect has been more closely associated with working on and thinking about contemporary Detroit's plight than Zago. In his latest project, he's created a bright, cheerful refuge in the city's desolate landscape. Read More
Real Estate Without Us (+Rendering)
Monday, February 23, 2009
Matt Chaban.
There have been countless symbols for the end of the real estate boom, both literal–the collapse of Countrywide, the Fannie & Freddie takeover, the unfinished tract homes and decaying "For Sale" signs–and figurative–the Eastside crane accidents, the TVCC Fire. But we think this back-to-nature scene spotted over the weekend in Williamsburg takes the, uh, mortgage. Read More
The Stimulus, Uploaded for Your Pleasure
Wednesday, February 18, 2009
Matt Chaban.
There has been a great deal of criticism that the Obama administration has failed to uphold a number of its campaign promises, such as shirking lobbyists and business-as-usual, and reaching across the aisle to craft bipartisan public policy. One thing that has not changed, however, is the commitment to open and transparent governance, particularly through the use of the World Wide Web. And so, today, Recovery.gov was launched, the better to help America keep tabs on the stimulus bill. Read More
On the Right Track?
Wednesday, February 18, 2009
Alan G. Brake.
Yesterday afternoon in Denver, Colorado, President Obama signed the stimulus bill into law. The process of doling out the spoils begins, as we wait, and hope, for the desired economic recovery. One piece of good news for urbanites and green transportation advocates, the bill includes $8 billion for high-speed rail, according to Politico. Read More
Stimulus and Sandwiches
Monday, February 16, 2009
Mariana Rodriguez Orte.
The second of the "Not Business as Usual" lunchtime conversation series last Wednesday at the Center for Architecture in New York had quite a turnout, including laid off designers, freelancers, and managers of struggling firms. Everyone was looking for ideas in these turbulent economic times. Read More
Big Turbine Keep On Turnin'
Thursday, January 15, 2009
Alan G. Brake.
Touchstone Architecture/Columbia River Crossing
Everyone seems to be talking about infrastructure and green jobs, which are expected to be a big part of any Federal stimulus package. One tension, however, is that a lot of infrastructure projects, especially highways, are anything but green. Here's one plan that attempts to reconcile this discrepancy, a wind turbine-equipped bridge planned for Portland, Oregon and Vancouver, Washington. Read More
The Downturn of the McMansion?
Tuesday, December 30, 2008
Alan G. Brake.
Amid the anxiety, speculation, and real hardship caused by the ongoing economic downturn, the provocative thesis of this Washington Post article stands out, which, if correct, could hold a silver lining for architects. Reporter Elizabeth Razzi interviews housing historian Virginia McAlester about how previous periods of economic declines shaped consumer demand for housing. The answer is simple and somewhat obvious: the demand for small houses rises. Her predictions for this cycle are less so. Read More
Not So Fast, Seaport Edition
Friday, December 19, 2008
Matt Chaban.
The news that General Growth Properties–which is on the verge of bankruptcy due to a massive debt-load related to its acquisition of the Rouse Company in 2004–put three historic properties up for sale has led some observers to speculate that development plans for one of them–New York's South Street Seaport–have hit the dustbin. Not so, AN has learned.
Strike Two? Not So Fast
Thursday, December 18, 2008
Matt Chaban.
First Laurie Olin, now Frank Gehry. That was the news earlier this week when the Wall Street Journal reported that the Santa Monica-based architect had laid off "more than two dozen" staffers involved with Bruce Ratner's Atlantic Yards project. What followed was a string of cheers predicting the troubled Brooklyn mega-development's demise. After all, how could it go on without its signature architect?
While considering this question, I kept thinking of a comment made by Kermit Baker yesterday, during an interview about the abysmal November billings index. Given what's going on elsewhere in the industry, the termination of a handful of architects may not signal the doomsday scenario the project's critics would like, and instead may be one more credit-related payroll pause like many others around the nation: Read More
Advertise on The Architect's Newspaper.
East Coast
West Coast


text:
So I learned Friday at daycare that my favorite teacher is leaving. She is leaving because she works an afternoon shift, is a college student, and she can't get her classes she needs outside of her shift. I talk to her every day and learned a couple of weeks ago that she's worked on and off at the center since 17. She is so loved by so many people. I would be standing there with Isaac, and these little kids would walk by, say by to her and give her hugs. It was just honestly the sweetest thing in the world.
I'm sad because she loved him. Took such good care of him. Worked on sitting, worked on teething and feeding with him. I met the new teacher Friday too (they train side by side for a week) and she did comment on what a great baby he was, but dammit I want Alyse to stay. She's the one that I've picked my son up from for 5 months, and answered my question, helped me troubleshoot feeding issues, took my feedback on helping with reflux. As much as she's been fantastic with Isaac, she's been equally good with me.
I had this issue too when they added a second afternoon teacher, becasue she didn't know Isaac! Now granted I've warmed up to her and Isaac loves her, and she loves Isaac (I pick up from the other afternoon teacher the other week and it was just the two of them, and they were on the floor playing and she gave him kisses).
I know its dorky, but you worry if this person will get to know my son, know his story, know his cues, pick him up while crying. Im going to cry Friday. I want to figure out someting awesome for her, cause honestly I don't know if I could have personally transitioned him to daycare without her.
Now the three remaining teachersr, the office staff, all love him. I even had someone from another room who had to go in there and cover a lunch break tell me she really liked Isaac ( I think she came form the one year old room which is nice too) and I'm sure the new teacher will love him too. Atleast I know in th emorning they still fight to recieve him (we pass over a door) and call him handsome and still threaten to take him.
I'm just a dork, sitting here about to cry (stupid Aunt Flo) just wanted to vent. It will be fine, and Alyse still wants to babysit for us. Isn't that great, we have no family here, and I always wondered who would babysit - who better than a former daycare teacher.


text: An investor looks through stock information at a trading hall in Haikou, the capital of Hainan province in southern China. Since mid-June, the main Shanghai stock index has lost 30 percent. Zhao Yingquan/Xinhua/Landov hide caption itoggle caption Zhao Yingquan/Xinhua/Landov The Two-Way China Takes Steps To Halt Plunge In Stock Markets July 5, 2015 The main Shanghai stock index has dropped 30 percent since mid-June, triggering margin calls that have fueled further selling.


text:
PEOPLE Gail Gonzales is joining Rodale on June 15th as VP, Associate Publisher in the Trade Books division, replacing Kristin Kiser. Gonzales has been at Simon & Schuster as Director of Integrated Marketing. Harper Children's President and Publisher Susan Katz is retiring after 30 years with the company. Suzanne Murphy has been hired as her successor. She…Continue Reading
July Posts
Subscribe to Email Updates
Follow Us
Upcoming EventsEvents on 07.07.201507.07.2015 – 07.12.2015Location: Grand Hyatt New York, 109 East 42nd Street, New York, NY 10017, United States ...More detailsEvents on 07.09.201507.09.2015 – 07.13.2015Location: San Diego Convention Center, 111 West Harbor Drive, San Diego, CA 92101, United States ...More detailsEvents on 07.17.201507.17.2015 – 07.19.2015Location: New York, NY ...More detailsEvents on 07.17.201507.17.2015 – 07.20.2015Location: Stokes Barn, Much Wenlock, Shropshire, TF13 6DB ...More detailsEvents on 07.18.201507.18.2015 – 07.21.2015Location: St Louis, MO, USA ...More details


text:
THIS IS 100% SARCASTIC I would love to see a make-up tutorial like this. Hope you guys had an awesome week. See you with a new video tommorow Jeffrey
Just to say happybirthday Josh :D Not the funniest but I wanted to get something up
This isn't my normal video, but its just a video I did cause I was bored:L Hope your well Jeffrey


text:
Environmental Assessments Companies in Ethiopia
Category: Environmental Management ×
Subcategory: Environmental Assessments ×
Location: Africa ×
Location: Ethiopia ×
Applied Technology & Management, Inc
based in West Palm Beach, FLORIDA (USA)
Applied Technology & Management (ATM) is a coastal, environmental, marine, and water resources engineering, design, and consulting firm serving public and private clients throughout the world. ATM has teams specialized in a number of niche services, including comprehensive marina services, boating infrastructure development, environmental, utilities, and coastal ...
Edinburgh Instruments Ltd
based in Livingston, UNITED KINGDOM
Edinburgh Sensors are proud to be global providers of high quality Gas Sensor solutions. Our diverse range of gas sensors use the latest technology to enable reliable, accurate and continuous gas detection. Established for over 20 years, Edinburgh Sensors use proven technology to deliver OEM Gas Sensors and Gas Monitors / Indoor Air Quality Monitors that are smart, ...
EnviroGulf Consulting
based in Fortitude Valley, AUSTRALIA
EGC is committed to providing professional, cost-effective services that are tailored to meet the specific needs of clients. The main objectives of the practice are to provide environmental management planning and specialised forms of consulting and technical services to private and public sector clients in the following fields: oil and gas industry, mining and minerals ...
Chubb Group of Insurance Companies
based in Warren, NEW JERSEY (USA)
Chubb Group of Insurance Cos. is a leading global property and casualty insurance organization. Since 1882, members of the Chubb Group of Insurance Companies have provided property and casualty insurance products to customers around the globe. These products are offered through a worldwide network of independent agents and brokers. The Chubb Group of Insurance Companies ...
based in Limonest, FRANCE
ACOEM helps customers in the industrial, environmental and defence sectors make the right decisions and take the right actions. To ensure the productivity and reliability of industrial machines. To prevent noise and vibration pollution. To protect personnel, sites and vehicles in military theaters of operation. To contribute to the development of effective, robust & ...
DNV GL - Business Assurance North America
based in Katy, TEXAS (USA)
As a world-leading certification body, we work with companies to assure the performance of their organizations, products, people, facilities and supply chains through certification, verification, assessment and training. As part of the larger DNV GL Group, our 2,000 Business Assurance employees worldwide help our customers build sustainable business performance and create ...
DelAgua Group
based in Marlborough, UNITED KINGDOM
The DelAgua kit was first introduced to overcome the problem of lack of access to testing laboratories. It is now used all over the World by both Government and Non-Government Organisations as the Portable Water testing kit of choice.DelAgua also supplies all manner of water testing & laboratory equipment at heavily discounted prices to companies and individuals all over ...
ETI Umwelttechnik AG
based in SWITZERLAND
ETI Umwelttechnik AG was founded in 1982 by ETI Group International with headquarters in Reno, Nevada / USA. In 1987 ETI (Europe) Ltd. became an independent Swiss company. ETI Environmental Technology International diversified its services, and moved from Landquart to own premises in Chur in 1991. For over 23 years, ETI has been concentrating its services on Persistent ...
based in Englewood, COLORADO (USA)
IHS is the leading source of information and insight in critical areas that shape today's business landscape – including environmental, health, safety and sustainability (EHS&S). Businesses and governments worldwide rely on IHS software, content and expertise to manage a broad spectrum of critical EHS business challenges, including operational risk, product stewardship, ...
based in Herndon, VIRGINIA (USA)
Since its foundation in 1901 as the Engineering Standards Committee, BSI Group has grown into a leading global independent business services organization providing standard-based solutions in more than 140 countries. BSI Group: develops private, national and international standards. certifies management systems and products. provides testing and certification of products ...


text:
Thanks for redeeming.{{else}}
Success! Thanks for repurchasing.{{/if}} {{else}} {{#if Expired}}
Increase my visibility!{{else}} {{#if IsMultiPack}}
{{#if HasMultiPack}} You have {{MultiPackCount}} top spot(s) left. Now loading you into the search listings... {{else}} You have used all top spots. Now loading you into the search listings... {{/if}}{{else}}
We are loading you into the search listing pages...{{/if}} {{/if}} {{/if}} {{/if}}
{{Current}} just saw you!{{/if}} {{#if LastTopSpotPurchaseIsMulti }} {{#if HasAnyTopSpot}}
{{#if HasFreeTopSpot }} {{ UnclaimedTopSpotFree }} Free {{/if}} {{#if HasPaidTopSpot }} {{#if HasFreeTopSpot }} & {{/if}} {{ MultiPackCount }} {{/if}} Top Spot{{#if Plural}}s{{/if}} Left{{else}}
0 Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{else}} {{#if HasFreeTopSpot }}
{{ UnclaimedTopSpotFree }} Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{/if}} {{else}} {{#if HasResults}}
{{Current}} see you now!{{else}} {{/if}} {{/if}}
You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
25 year old woman | Jacksonville, FL, USA | Seeking men 24-29 within 25 miles
Tired of Mr. Wrong and ready for Mr. Right
I am looking for someone to make me laugh and be there when I need to vent about anything someone who I can depend on, and who can cheer me up when I am feeling blue. I am looking to find someonw who wants to travel with me and try new things. I am tired of having my heart broken by the wrong guy and im ready to find someone who can appreciate the little things. I am someone who is always there when my friends need me and I am a problem solver. I never give up on anything and always finish. So feel free to message me
Coffee and conversation , Cooking , Dining out , Movies/Videos , Music and concerts , Exploring new areas , Nightclubs/Dancing , Playing cards , Travel/Sightseeing , Wine tasting
Billiards / Pool, Bowling, Dancing, Football, Golf, Other types of exercise, Swimming, Yoga
I exercise 5 or more times per week.
I like Cats, Fish
Im not a big pet person, I have allergies for pets with a thick fur coat.
I love traveling! I go to Orlando a lot, and I like driving down south to the Miami area. I am originally from Canada so I make a trip at least once a year.
University of North Florida, Jacksonville, FL
I am a TV Junkie, at my house they call me the TV guide. I love ABC shows, my newest favorite is Big Bang Theory and Mad Men. When it comes to music I like to listen to a little of everything, and I love cooking and trying new foods.
I enjoy going to the movies, and love going to the beach in the summer. There is not much to do in Jacksonville so I love going to new places.
The last book I read was the Hunger Game novels. I finished all three in a week, and cannot wait to see the movies. I love reading I am a fast reader and anything that sounds good I will give it a try.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'2" (157cms)
|5'6" (167cms) to 6'0" (182cms)
|Body type:
|No preference
|No preference
|Dark brown
|Black, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way, Occasionally, Daily
|Social Drinker
|Social Drinker
|Executive / Management
|No preference
I currently work at Bank of America as a supervisor in a call center. I am the person you speak to when you ask for a manager. I do plan on working my way up the executive ladder, and plan on working in management.
|$35,001 to $50,000
|$35,001 to $50,000
|Never Married
|Never Married
|Have kids:
|Wants kids:
|East Indian
|Asian, East Indian, Latino / Hispanic, Middle Eastern, White / Caucasian
|No preference
|Bachelors degree
|Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral
I just recently graduated from the University of North Florida with my Bachelors in International Business, I do plan on returning to school to get my MBA


text:
Article ID: 231413 - View products that this article applies to.
This article was previously published under Q231413
Moderate: Requires basic macro, coding, and interoperability skills.
This article applies to a Microsoft Access database (.mdb) and to a Microsoft Access project (.adp).
On This Page
If you have not set the appropriate references in Access 2000, you may receive one of the following error messages when you run code that includes an object from a missing reference:
Variable not defined.
Run-time error '5':
Invalid procedure call or argument.
Microsoft Visual Basic
The library which contains this symbol is not referenced by the current project, so the symbol is undefined. Would you like to add a reference to the containing library now?
Compile Error: Can't find project or library.
Your database contains a reference to a database, a type library, or an object library that is marked as MISSING: <referencename> in the References dialog box.
Be sure that you include all required references. To check for and remove missing references:
The references that are selected by default at the time a new Microsoft Access 2000 database is created are as follows:
Steps to Reproduce the Behavior
Article ID: 231413 - Last Review: June 28, 2004 - Revision: 2.0
Give Feedback


text:
Oracle and shmmax and shmall
|Posted by Sonia Hamilton under Oracle
A good article on Oracle and shmmax and shmall. In a nutshell:
- shmall it is not the maximum amount of memory which can be allocated, it is the maximum size of any shared memory chunk – how big a bite you want per bite from free memory.
- shmmax – its value determines the maximum amount of memory that ALL shared memory can take. the actual setting is derived… the maximum amount of memory = shmall * pagesize where pagesize = getconf PAGE_SIZE and shmall = cat /proc/sys/kernel/shmall
Share This
One Response to Oracle and shmmax and shmall
Handy if you mix up shmall and shmmax..


text:
BEIRUT: In the hundred odd years since the first Danish film – photographer Peter Elfelt's 1897 one-minute documentary "Travelling with Greenlandic Dogs" – Danish cinema has come on in leaps and bounds.
This week the Metropolis Cinema Sofil is celebrating the diverse achievements of Denmark's directors with a seven-night festival entitled "Danish feast: A taste of classic and contemporary film makers."
The selection is eclectic, spanning a wide range of genres and several decades, though for the most part serving up one of the better known films from each of the seven directors showcased. The lineup includes three classic films, two contemporary numbers and two feature documentaries.
The festival kicks off with part of a trilogy by Lars von Trier, famous for his avant-garde technique and his work on the Dogme 95 movement which aimed to simplify filmmaking. The film on show is not Von Trier's most recent offering, "Melancholia," released last year, but a far older work – the final film in his Europe trilogy, "Europa" (1991).
Set in Germany, just after World War II, "Europa" follows Leo (Jean-Marc Barr), a young American of German descent, when he takes a job on a sleeper train. Leo tries to remain neutral but soon finds himself caught between the Americans and an underground Nazi gang known as the Werewolves. When Leo falls in love with the train company owner's daughter, he is faced with some difficult choices.
Thursday night brings us forward in time to 2010 with "Submarino," a tale of two estranged brothers directed by Thomas Vinterberg, a co-founder of the Dogme 95 movement, along with Von Trier.
"Submarino" explores family relationships through the story of Nick (Jakob Cedergren) and his little brother, raised by their alcoholic mother and divided by tragedy. Years later, Nick – now an alcoholic ex-con – and his nameless younger brother – a junkie single-parent who deals drugs for the money to feed his son – meet again.
Friday's showing promises to inject some political history into the weekend with "President," Christoffer Gulbrandsen's 2011 documentary about the reluctant first president of Europe, related by current and former heads of state. Gulbrandsen described the film as an insight into "a humorous and grotesque world," likening it to an "academic reality TV show."
Saturday's feature moves from contemporary back to classic with director Gabriel Axel's best-known film, the Oscar-winning "Babette's Feast" (1987), based on a novel by Isak Dinesen. Set in 19th century Denmark, the film recounts the tale of two adult sisters who live with their father, the village pastor.
The pair agrees to take in a French refugee, Babette, who is fleeing the 1871 Commune of Paris uprising. When Babette comes into some money she insists on cooking up a lavish feast for the puritanical sisters and a group of amazed local villagers.
The Cannes Grand Prize winning documentary "Armadillo" (2010) by Janus Metz will be playing Sunday – a war drama shot over a period of six months in Helmand, a province in southern Afghanistan. Metz traveled to the region with a group of Danish soldiers, repeatedly risking his life to film gun battles between Danish and Taliban troops.
The result is a tense, insightful exploration of the psychology of warfare, which charts the increasing cynicism, paranoia and adrenaline addiction among the young Danish soldiers slated to help the Afghan troops. The film provoked furious debate in the filmmaker's homeland as a result of the behavior of the soldiers it documents.
A true classic is scheduled for Monday night. The oldest film included in the program, Henning Carlsen's black and white feature "Hunger" (1966) is a socio-realistic drama about Pontus (Per Oscarsson), a penniless poet.
Based on a novel by Knut Hamsun, the plot centers around Pontus' unfortunate combination of pride and poverty, which renders him desperate for money but unwilling to accept an advance from the newspaper to which he wishes to submit an article.
Set in Oslo in 1890, the film delves into the mental world of the starving young poet. Evicted from his home he wanders the streets, his hunger causing him to lose track of what is real and what is imaginary, until the boundaries between fact and fiction become little more than a blur.
The festival closes on Oct. 2 with Nicolas Winding Refn's "Valhalla Rising" (2010). One of Refn's more recent films – though set in the year 1000 A.D. – the plot centers on One Eye, a mute Viking warrior with supernatural strength.
One Eye is being held captive by Barde, a Norse chieftain, but escapes with the help of Are, a boy slave. The two sail away on a Viking ship into the darkness of a mysterious mist only to find, when it clears, that they have reached a new world where a bloody fate awaits them.
The wide range of films on show should ensure that there is something to appeal to every palate. Film buffs will find the selection overall a comprehensive introduction to seven of Denmark's key directors and a fitting tribute to Danish cinema's output over the past three decades.
"Danish Feast: A taste of classic and contemporary film makers" is running at Metropolis Empire Sofil until Oct. 2. All films are scheduled to begin at 8 p.m., cost LL5,000 and feature English audio or subtitles. For more information, please visit http://www.metropoliscinema.net/
Your feedback is important to us!
We invite all our readers to share with us their views and comments about this article.
Disclaimer: Comments submitted by third parties on this site are the sole responsibility of the individual(s) whose content is submitted. The Daily Star accepts no responsibility for the content of comment(s), including, without limitation, any error, omission or inaccuracy therein. Please note that your email address will NOT appear on the site.
Alert: If you are facing problems with posting comments, please note that you must verify your email with Disqus prior to posting a comment. follow this link to make sure your account meets the requirements. (http://bit.ly/vDisqus)
Visions of a public Dalieh
Sidon archaeological site alters global views
Capturing consumption on camera


text:
'Hayden' Ruffle Racerback Underwire Foam Push-Up Bra
|Item # 646885
Details & Care
Flirty ruffles and jacquard-dot mesh fashion the flirty racerback of an underwire bra created with cleavage-enhancing padded foam cups. Metallic fibers make the straps and underbust band shine.
- Neon colors are brighter in person.
- Front magnetic closure.
- Lined.
- 84% nylon, 16% spandex.
- Hand wash cold, line dry.
- By kensie; imported.
- Lingerie.
Watch the video: Bra Fit Video
Learn More: Bra Fit Guide


text:
Jump to content
Remember me
This is not recommended for shared computers
Sign in anonymously
Don't add me to the active users list
Banner advertising on our site currently available from just $5!
1. Learn about the promo. 2. Sign up for GDNet+. 3. Set up your advert!
17 October 2014 - 01:30 AM
Design an AI that plays the game first. Until you do that, there is no "learning" that can happen.
Design an AI that plays the game first. Until you do that, there is no "learning" that can happen.
Ok, but I'm doing some research now so I'll know what I can and can't do.
17 October 2014 - 01:28 AM
Details about the features you intend to implement.
It's supposed to be a normal strategy game with some extra features...
for example the player would be able to build a bridge in order to mobilize its units to areas it couldn't access without it.
I'd like the AI to know that unless the user shows signs of building that bridge, it won't build up it defences in that area or if the user shows interest in some region, the AI will be ready for him.
16 October 2014 - 11:16 AM
what do you mean by "game design"?
19 May 2013 - 02:32 PM
I tried using Waitable Timers like in this example:
But it seems that the event is signaled only after 1 milisecond and after not the time I specified...
I tried using:
double timeoutMicro = 400;
liDueTime.QuadPart = -1 * timeoutMicro * 10;
19 May 2013 - 10:10 AM
@Hodgman & galop1n:
I'm trying to sample another process (memory, stack, etc) and I'm checking the boundaries of what I can and cannot do (without using all the resources of the computer).
GameDev.net™, the GameDev.net logo, and GDNet™ are trademarks of GameDev.net, LLC