FreshPatents.com Logo
stats FreshPatents Stats
n/a views for this patent on FreshPatents.com
Updated: April 14 2014
newTOP 200 Companies filing patents this week


    Free Services  

  • MONITOR KEYWORDS
  • Enter keywords & we'll notify you when a new patent matches your request (weekly update).

  • ORGANIZER
  • Save & organize patents so you can view them later.

  • RSS rss
  • Create custom RSS feeds. Track keywords without receiving email.

  • ARCHIVE
  • View the last few months of your Keyword emails.

  • COMPANY DIRECTORY
  • Patents sorted by company.

AdPromo(14K)

Follow us on Twitter
twitter icon@FreshPatents

Thrombospondin fragments and uses thereof in clinical assays for cancer and generation of antibodies and other binding agents

last patentdownload pdfdownload imgimage previewnext patent


20120276559 patent thumbnailZoom

Thrombospondin fragments and uses thereof in clinical assays for cancer and generation of antibodies and other binding agents


The invention relates to thrombospondin fragments found in plasma, their use or use of portions thereof in diagnostic methods, as method calibrators, method indicators, and as immunogens, and as analytes for methods with substantial clinical utility; and their detection in plasma or other bodily fluids for purpose of diagnostic methods, especially for cancer.
Related Terms: Thrombospondin

Browse recent W2 Holdings, Inc. patents - Philadelphia, PA, US
Inventor: Kevin J. Williams
USPTO Applicaton #: #20120276559 - Class: 435 792 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >Assay In Which An Enzyme Present Is A Label >Heterogeneous Or Solid Phase Assay System (e.g., Elisa, Etc.)

view organizer monitor keywords


The Patent Description & Claims data below is from USPTO Patent Application 20120276559, Thrombospondin fragments and uses thereof in clinical assays for cancer and generation of antibodies and other binding agents.

last patentpdficondownload pdfimage previewnext patent

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application 60/405,494 filed Aug. 23, 2002; U.S. application Ser. No. 10/419,462, filed Apr. 21, 2003; and PCT application PCT/US03/26023, filed Aug. 20, 2003.

FIELD OF THE INVENTION

The present invention relates to assays for blood levels of one or more thrombospondin fragments as a diagnostic test for cancers and other diseases, the use of such fragments and/or derivatives thereof to generate specific antibodies and other binding agents and/or to use as calibrators, competitors, and/or indicators in an assay, and to the fragments themselves. Specifically, the present invention relates to specific detection of high molecular weight fragments and forms of thrombospondin in cancer patients as compared to non-cancerous patients. The present invention also relates to methods of distinguishing between proper and improper sample collections of plasma as indicated by the detection of high molecular weight thrombospondin fragments and/or thrombospondin itself.

BACKGROUND OF THE INVENTION

Thrombospondin (TSP), also known as TSP-1, is a multimeric glycoprotein comprised of identical monomers. The monomers migrate at an apparent molecular weight of approximately 185 kDa in SDS-polyacrylamide electrophoretic gels under reducing conditions. The predominant multimer is a trimer, which migrates at an apparent molecular weight of approximately 450 kDa on non-reducing gels. The molecular weights by sedimentation equilibrium are similar, at 135 kDa for monomers and 420 kDa for trimers. The predicted molecular weight from just the sequence of amino acyl residues in the monomer is 127,524 Da, which does not include contributions from glycosylation and β-hydroxylation. The thrombospondin glycoprotein is produced by platelets and is released upon platelet activation from platelet α-granules, along with many other proteins, such as platelet-derived growth factor, β-thromboglobulin, fibronectin, fibrinogen, and platelet factor-4 (see Chapter 1, “An introduction to the thrombospondins” in The Thrombospondin Gene Family by J C Adams, R P Tucker, & J Lawler, Springer-Verlag: New York, 1995, pp. 1-9, but especially p. 2; and Chapter 3, “The secondary and tertiary structure of the thrombospondins,” ibidem pp. 43-56, especially Table 3.1). Thrombospondin is known to be involved in biological processes such as cell adhesion, proliferation and chemotaxis. It has also been reported that thrombospondin may be involved in the progression of malignant tumors. Furthermore, thrombospondin has been reported to be highly expressed in many human malignant tissues and in surrounding stroma and/or endothelium and has been reported to be present in higher than normal levels in the plasma of cancer patients. (e.g., Qian and Tuszynski, Proc. Soc. Exp. Biol. Med., 212:199-207, 1996; de Fraipont F et al. Trends Mol. Med., 7:401-407, 2001).

Despite the foregoing, as for any potential diagnostic test, it would be desirable to increase the specificity and sensitivity of such tests. To that end, the present inventor has discovered that thrombospondin is present in the blood and blood plasma in relatively small amounts compared to fragments of thrombospondin, and this finding is true in the blood and blood plasma of cancer patients as well. This discovery provided a basis for the present inventions related to novel diagnostic assays that are more specific, more sensitive, more easily calibrated, and in some cases distinguish these thrombospondin fragments from each other and from thrombospondin itself. The present inventor has also identified specific high molecular weight thrombospondin fragments, forms, and/or cross-reacting material is useful in the detection of cancer. The present invention also relates to novel methods of distinguishing between a properly versus an improperly collected sample by detecting high molecular weight thrombospondin fragments, forms, cross-reactive material, and/or thrombospondin itself.

BRIEF

SUMMARY

OF THE INVENTION

Important aspects of the invention are diagnostic methods and related kits that are based on the detection and quantification of thrombospondin fragments and/or thrombospondin in bodily fluids, especially plasma. Foremost among those diagnostic methods are those that detect or monitor the status of a cancer.

Aspects of the invention closely related to the diagnostic methods are thrombospondin fragments that are detected in the plasma, thrombospondin fragments that can be used to induce an antibody of interest for use in a diagnostic method or can be used in a competition-type or non-competitive diagnostic assay. Another important aspect of the invention includes the detection of high molecular weight thrombospondin fragments in cancerous versus non-cancerous plasma samples. Aspects of the invention also relate to methods of assaying proper sample collection by analysis of high molecular weight thrombospondin fragments and/or thrombospondin itself. The sample includes but is not limited to blood, serum or plasma.

Thrombospondin Fragments of the Invention

In one aspect, the invention is a purified thrombospondin fragment that has been extracted from a bodily fluid, especially plasma, said fragment being one within a molecular weight range selected from the group consisting of 80 to 148 kDa, 40 to 64 kDa, and 22 to 36 kDa, wherein the size in kDa is the apparent size on gel electrophoresis after disulfide bond reduction. Their uses include, but are not limited to, a) the induction of an antibody and/or other binding agent of interest, b) induction of an antibody and/or other binding agent for a diagnostic method, c) use in a competition-type diagnostic assay, d) as a reference molecule in an assay for a thrombospondin fragment or fragments or thrombospondin of human subjects, and e) the immunization of an animal. In a closely related aspect, the invention is a polypeptide or modified polypeptide, made by recombinant and/or chemical techniques, that has the identical primary structure as one of said purified thrombospondin fragments or a portion thereof. Such chemical techniques include, but are not limited to, glycosylation, β-hydroxylation, alkylation and reduction.

In particular embodiments, the fragment\'s molecular weight is one within a molecular weight range selected from the group consisting of 80 to 148 kDa, 40 to 64 kDa, and 22 to 36 kDa. Specific examples of fragment molecule weights are 85, 90, 50, and 30 kDa. Preferably, the fragment is one found in human plasma.

In a related aspect, the invention is a purified and/or synthetic thrombospondin fragment or portion thereof, said fragment being one that starts between amino acid I-165 (just after the N12/I peptide) and V-263 (the start of the procollagen homology domain), inclusive (i.e., inclusive of I-165 and V-263), and ends between amino acid K-412 (the end of the reported collagen type V-binding region) and I-530 (the end of the domain of type 1 repeats), inclusive. Preferred are such fragments that start between N-230 and G-253, inclusive (at or near the start of the domain of interchain disulfide bonds, I-241, which is the first residue downstream [meaning towards the C-terminus of the full protein] of a predicted cleavage site for chymotrypsin and/or a chymotrypsin-like protease), and end at between V-400 and S-428, inclusive (at or near a predicted chymotrypsin cleavage site, F-414, that falls two residues after the end of the collagen type V-binding region), said portion being at least 3 amino acyl acids in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues).

In a further related aspect, the invention is a purified and/or synthetic thrombospondin fragment or portion thereof, said fragment being one that starts between amino acid I-165 (just after the N12/I peptide) and V-263 (the start of the procollagen homology domain), inclusive, and ends between amino acid I-530 (the end of the type 1 repeats) and R-733 (the end of the first type 3 repeat), inclusive. Preferably such a fragment starts between N-230 and G-253, inclusive, and ends between D-527 and S-551, inclusive, which is at or near a predicted chymotrypsin cleavage site, F-539, in the first type 2 repeat; said portion being at least 3 amino acyl acids in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues).

In a still further related aspect, the invention is a purified and/or synthetic thrombospondin fragment or portion thereof, said fragment being one that starts between amino acid I-165 (just after the N12/I peptide) and V-263 (the start of the procollagen homology domain), inclusive, and ends between amino acid R-792 (the end of the third type 3 repeat) and Y-982 (the third of the predicted chymotrypsin cleavage sites in the C-terminal domain), inclusive. Preferably such a fragment starts between N-230 and G-253, inclusive, and ends between G-787 and V-811, inclusive, which is at or near a predicted chymotrypsin cleavage site, Y-799, in the fourth type 3 repeat; said portion being at least 3 amino acyl acids in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues). Protein molecular weights here were computed using standard computational aids (such aids are available, for example, at the web site of the Bioinformatics Organization, Inc., http://bioinformatics.org/sms/prot_mw.html; see Stothard, P. 2000. The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. BioTechniques 28: 1102-1104) and adjusted upwards to account for post-translational modifications. Predicted cleavage sites for chymotrypsin (and any closely related protease) were identified using tools available from the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB) (See http://us.expasy.org/cgi-bin/peptidecutter/peptidecutter.pl) and were limited to predicted sites of at least 80% probability. The uses of said fragments and portions include, but are not limited to, the induction and/or screening of an antibody and/or another binding agent of interest in a diagnostic method and use in a diagnostic assay. In particular embodiments, the invention is one of the specified fragments, rather than a portion thereof. In additional embodiments, a fragment and/or a portion can incorporate or be linked to a label and/or a carrier.

Throughout, wherever reference is made to a fragment or a portion thereof (or an immunoreactive portion thereof), it is understood that the fragment is a preferred embodiment of the invention. It is also understood throughout this Application that immunogenic portions, immunoreactive portions, cross-reactive portions, and/or epitopes are generally six amino acyl residues long or longer, but an occasional portion or epitope can be shorter. Such shorter portions or epitopes are also contemplated.

Six additional aspects are:

1) A purified and/or synthetic thrombospondin fragment, said fragment being at least 6 contiguous amino acyl residues in length, and wherein the fragment comprises a protease-resistant core domain or a part thereof, said domain or part thereof being selected from the group consisting of a domain of inter-chain disulfide bonds, an oligomerization domain, a procollagen-like domain, a type 1 repeat, a type 2 repeat, and a type 3 repeat, said part being at least 6 amino acyl residues in length.

2) A purified and/or synthetic thrombospondin fragment, said fragment being at least 6 contiguous amino acyl residues in length, and wherein the fragment comprises an amino acid sequence selected from the group consisting of TEENKE (SEQ ID NO:1), CLQDSIRKVTEENKE (which includes an N-terminal Cys added to aid conjugation) (SEQ ID NO:2), LQDSIRKVTEENKE (SEQ ID NO:3), EGEARE (SEQ ID NO:4), PQMNGKPCEGEARE (SEQ ID NO:5), EDTDLD (SEQ ID NO:6), YAGNGIICGEDTDLD (SEQ ID NO:7), CNSPSPQMNGKPCEGEAR (SEQ ID NO:8), RKVTEENKELANELRRP (SEQ ID NO:9), CRKVTEENKELANELRRP (which includes an N-terminal Cys added to aid conjugation) (SEQ ID NO:10), PQMNGKPCEGEAR (SEQ ID NO:11), CEGEAR (SEQ ID NO:12), and RKVTEENKE (SEQ ID NO:13). (In particular embodiments the fragment comprises two, or even all of the foregoing sequences).

3) a purified and/or synthetic thrombospondin fragment, said fragment being at least 6 contiguous amino acyl residues in length, and wherein the fragment comprises a collagen type V binding domain or a portion thereof.

4) A purified and/or synthetic thrombospondin fragment, said fragment being at least 6 contiguous amino acyl residues in length, and wherein the fragment comprises an epitope for binding at least one of the following commercially available antibodies, each of which recognizes a ˜450 kDa (non-reduced) protein that is specifically identified as thrombospondin (the TSP Ab numbering, e.g., “TSP Ab-2”, comes from Lab Vision Corporation, Fremont, Calif., which currently has a web site at http://www.labvision.com/; clone designations refer to the hybridoma clone that produces a particular monoclonal antibody) It is also understood that said fragment includes a fragment that can be designed to bind a pre-existing monoclonal antibody, through the use of peptide scanning analysis, competition experiments, and other methods known in the art. It is also understood that the current invention includes, but is not limited to, uses of pre-existing antibodies independent of a purified and/or synthetic fragment, some of which uses are also listed below.

TSP Ab-2 (Clone D4.6):

This antibody is stated to react against reduced and non-reduced protein, and its epitope has been reported to be in the calcium-binding domain of TSP (C-terminal 50 kDa piece of the 120 kDa fragment from protease digestion of Ca-replete TSP). The calcium-binding region is generally considered to be in the type 3 repeats (TSP residues 698-925). For example, it is expected that TSP Ab-2 will bind thrombospondin but not the 30 kDa circulating fragment. This antibody can be used to detect and/or quantify TSP and/or a circulating fragment; distinguish thrombospondin from a circulating fragment; and/or distinguish one or more fragments from each other. It has been reported to show no cross-reaction with fibronectin, fibrinogen, or von Willebrand factor. Its binding to thrombospondin is enhanced by EDTA i.e. at low [Ca2+]. Applicant\'s data indicates that this antibody also binds three major fragments (of apparent molecular weights of ˜30 kDa, ˜50 kDa, and ˜115 kDa) of TSP in human (and dog) plasma, and several minor fragments of TSP in human (and dog) plasma, as well as high molecular weight fragments or forms (above the ˜115 kDa band) (See FIG. 5).

TSP Ab-4 (Clone A6.1):

This antibody is stated to react against reduced and non-reduced protein, and its epitope has been reported to be in the collagen type V-binding domain. This antibody binds thrombospondin, and the applicant has discovered that it binds the three major TSP fragments in human plasma. Thus, this antibody can be used to detect and/or quantify TSP and/or a circulating fragment or fragments. In combination with another antibody or binding agent, it can be used in an assay to distinguish thrombospondin from a circulating fragment; and/or to distinguish one or more fragments from each other. Applicant\'s data shows that this antibody also binds three major (˜30 kDa, ˜50 kDa, and ˜115 kDa) and several minor fragments of TSP in human (and dog) plasma, as well as several molecular weight fragments or forms (above the ˜115 kDa band) (See FIGS. 3, 4 and 6). This antibody, which is a mouse monoclonal, can be used in sandwich ELISAs for capture or detection and in competitive ELISAs (See FIG. 12). As an example meant to be illustrative and not restrictive, TSP Ab-4 is used to capture TSP and circulating fragments, and then the other antibody or binding agent is used for detection, but is able to distinguish TSP from a fragment or fragments, or one fragment from another. It is understood that TSP Ab-4 also binds thrombospondin and thrombospondin fragments from important non-human sources as well, including but not limited to the dog. Thus, the use of this antibody and/or a similar binding agent in an assay for a thrombospondin fragment or fragments in a sample from a non-human source, such as dog, is contemplated. This antibody shows no cross-reaction with fibronectin, fibrinogen, and von Willebrand factor. This antibody inhibits thrombospondin-collagen interaction, and its binding to thrombospondin is unaffected by glycosaminoglycans (e.g. hyaluronic acid, chondroitin sulfate, and heparin). Also, its binding is enhanced by EDTA i.e. at low conc. of Ca2+.



Download full PDF for full patent description/claims.

Advertise on FreshPatents.com - Rates & Info


You can also Monitor Keywords and Search for tracking patents relating to this Thrombospondin fragments and uses thereof in clinical assays for cancer and generation of antibodies and other binding agents patent application.
###
monitor keywords



Keyword Monitor How KEYWORD MONITOR works... a FREE service from FreshPatents
1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored.
3. Each week you receive an email with patent applications related to your keywords.  
Start now! - Receive info on patent apps like Thrombospondin fragments and uses thereof in clinical assays for cancer and generation of antibodies and other binding agents or other areas of interest.
###


Previous Patent Application:
Marker protein for type-2 diabetes
Next Patent Application:
5.9 kda peptide immunoassay method
Industry Class:
Chemistry: molecular biology and microbiology
Thank you for viewing the Thrombospondin fragments and uses thereof in clinical assays for cancer and generation of antibodies and other binding agents patent info.
- - - Apple patents, Boeing patents, Google patents, IBM patents, Jabil patents, Coca Cola patents, Motorola patents

Results in 0.72482 seconds


Other interesting Freshpatents.com categories:
Electronics: Semiconductor Audio Illumination Connectors Crypto ,  -g2-0.1977
     SHARE
  
           

FreshNews promo


stats Patent Info
Application #
US 20120276559 A1
Publish Date
11/01/2012
Document #
13290296
File Date
11/07/2011
USPTO Class
435/792
Other USPTO Classes
436501
International Class
01N33/566
Drawings
13


Thrombospondin


Follow us on Twitter
twitter icon@FreshPatents