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

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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.)

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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.

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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+.

TSP Ab-5 (Clone B5.2):

This antibody has been reported to react against reduced and non-reduced protein, and its epitope is in a 10 kDa fragment present at the junction of type 2 and type 3 repeats. The junctional region is listed elsewhere as residues 674-697, but this is only 24 residues and less than 10 kDa, so the epitope is less precisely mapped. It is expected that this antibody will bind TSP but not the 30 kDa circulating fragment; however, Applicant\'s data suggests that this antibody binds all three major fragments, including the −30 kDa fragment(s) 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. 7). Thus, this antibody can be used to detect and/or quantify TSP and/or a circulating fragment or fragments; distinguish thrombospondin from a circulating fragment; and/or distinguish one or more fragments from each other. It shows no cross-reaction with fibronectin, fibrinogen, and von Willebrand factor.

TSP Ab-9 (Clone MBC 200.1):

This antibody has been reported to react against reduced and non-reduced protein, and its epitope is in the N-terminal heparin-binding domain of thrombospondin. Thus, it should bind to thrombospondin but not to major circulating fragments. In Western blotting, Ab-9 reacts with a 25 kDa peptide (heparin-binding domain) from thermolysin digests of thrombospondin that is not disulfide bonded to any other region of the thrombospondin molecule. Heparin efficiently inhibits the binding of Ab-9 to thrombospondin. Thus, this antibody can be used to detect and/or quantify TSP; and/or distinguish thrombospondin from a circulating fragment or fragments. This antibody is not suitable for detecting all major fragments in the circulation.

TSP Ab-8 (Rabbit ws3 Antibody):

Recognizes a ˜450 kDa (non-reduced) or 180 kDa (reduced) protein, identified as TSP. This antibody, which is a rabbit polyclonal, can be used in sandwich ELISAs for capture or detection and in competitive ELISAs (see FIG. 12). Applicant has discovered that it binds the three major TSP fragments in human plasma (˜30 kDa, ˜50 kDa, and ˜115 kDa), and minor fragments of TSP in human (and/or dog) plasma, as well as its binding to larger fragments or forms (above the ˜115 kDa band) (See FIG. 9). 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.

As an example meant to be illustrative and not restrictive, one takes the difference between (a) the result of an assay using an antibody or binding agent that binds TSP and circulating fragments in plasma, versus (b) the result of an assay using an antibody or binding agent that binds TSP but not fragments in plasma. The antibody or binding agent in (a) is selected from the group consisting of TSP Ab-4, TSP Ab-8, TSP Ab-11, and an antibody or binding agent that binds TSP and circulating fragments in plasma. The antibody or binding agent in (b) is selected from the group consisting of TSP Ab-3, TSP Ab-6, TSP Ab-9, and an antibody or binding agent that binds TSP but not circulating fragments. Said assay in (a) detects TSP plus fragments; said assay in (b) detects TSP; said difference, (a) minus (b), thereby gives a quantification of fragments without TSP. Likewise, differences can be taken between (c) the result of an assay using an antibody or binding agent that binds TSP and a subset of the circulating fragments in plasma and/or serum, versus the result of (a), above, to obtain a quantification of the fragment or fragments not detected in (c). Differences can also be taken of the result of (c) versus (b), above, to obtain a quantification of the fragment or fragments detected in (c) but without the signal from TSP. The antibody or binding agent in (c) is selected from the group consisting of TSP Ab-2, TSP Ab-5, TSP Ab-1, TSP Ab-7, and an antibody or binding agent that binds TSP and only a subset of the circulating fragments.

TSP Ab-11 (Clones D4.6+A6.1+MBC 200.1):

The Ab-11 cocktail is designed for sensitive detection of thrombospondin by Western blotting. This antibody cocktail has been reported to show no cross-reaction with fibronectin, fibrinogen, or von Willebrand factor. Because it is a mixture of TSP Ab-2, TSP Ab-4, and TSP Ab-9, it detects TSP and 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. It can also be used in an assay for TSP and/or a TSP fragment or fragments in a sample from a non-human source, such as a dog.

Other antibodies that are useful, even though they have been disclosed only as binding non-reduced protein include, but are not limited to TSP Ab-1, TSP Ab-3, TSP Ab-6, and TSP Ab-7, which are described in more detail immediately below. The Applicant\'s results indicate that Ab-7 binds reduced material (See FIG. 8).

TSP Ab-1 (Clone A4.1):

This antibody has been reported to bind the N-terminal half of the central stalk-like region of thrombospondin. This region is recovered as a 50 kDa fragment after chymotryptic digestion of thrombospondin. Thus, Ab-1 may be used to detect and/or quantify TSP and/or a circulating fragment or fragments; distinguish thrombospondin from a circulating fragment or fragments; and/or distinguish one or more fragments from each other. TSP Ab-1 has been reported to show no cross-reaction with fibronectin, fibrinogen, and von Willebrand factor. It inhibits the adhesion of human melanoma G361 cells, keratinocytes, squamous carcinoma cells, and rat smooth muscle cells to thrombospondin. It does not inhibit aggregation of thrombin-induced platelets. This antibody is stated to block the anti-angiogenic activity of thrombospondin by inhibiting its binding to TSP-Receptor/CD36.

TSP Ab-3 (Clone C6.7):

This antibody has been reported to bind the platelet or cell-binding domain at the extreme C-terminus of TSP and should therefore distinguish TSP from fragments, as well as a fragment or fragments with vs. without this epitope. Thus, this antibody can be used to detect and/or quantify TSP; and/or distinguish thrombospondin from a circulating fragment or fragments; and/or distinguish a fragment from another. This antibody should not be suitable for detecting all of the three major fragments in the circulation, although it may detect higher molecular weight fragments or forms. Heparin or EDTA may marginally affect binding of Ab-3 to thrombospondin. Ab-3 blocks thrombospondin-mediated agglutination of fixed red blood cells. It shows no effect on thrombospondin-mediated agglutination of fixed, activated platelets. It inhibits both thrombin- and A23187-induced aggregation of washed, live (not fixed) platelets without affecting the secretion of serotonin. Ab-3 inhibits adhesion of melanoma G361 cells to thrombospondin, and blocks the binding of C-terminal domain to Integrin-Associated Protein (IAP)/CD47.

TSP Ab-6 (Clone A2.5):

This antibody has been reported to immunoprecipitate thrombospondin. This antibody has been reported to show no cross-reaction with fibronectin, fibrinogen, or von Willebrand factor. Its epitope has been reported to be localized in the heparin-binding domain of thrombospondin, and therefore, heparin efficiently inhibits the binding of Ab-6 to thrombospondin. Thus, this antibody can be used to detect and/or quantify TSP; and/or distinguish thrombospondin from a circulating fragment or fragments, as well as a fragment or fragments with vs. without this epitope. This antibody should not be suitable for detecting all of the three major fragments in the circulation, although it may detect higher molecular weight fragments or forms. Hyaluronic acid and chondroitin sulfate show no inhibition at low concentration and only partially inhibit over the concentration range at which heparin abolishes the binding. Thrombospondin binds with high affinity to a sulfated glycolipid or sulfatide found on red cell and platelet membranes. Ab-6 has been reported to block the binding of thrombospondin to sulfatides at low concentrations. Ab-6 has been reported to immunoprecipitate a 25 kDa peptide (heparin-binding domain) from chymotryptic digests of thrombospondin that is not disulfide bonded to any other region of the thrombospondin molecule. This antibody inhibits the hemagglutination of trypsinized, glutaraldehyde-fixed human erythrocytes by purified thrombospondin. It also inhibits the agglutination of fixed, activated platelets by thrombospondin. It does not inhibit either thrombin- or A23187-induced aggregation of washed, live platelets. Ab-6 does not bind to reduced and alkylated thrombospondin or thrombospondin transferred to nitrocellulose membrane after SDS-PAGE.

TSP Ab-7 (Clone HB8432):

This antibody has been reported to bind type 2 repeats. Thus, Ab-7 may be used to detect and/or quantify TSP and/or a circulating fragment or fragments; distinguish thrombospondin from a circulating fragment or fragments; and/or distinguish one or more fragments from each other. It shows no cross-reaction with fibronectin or any other serum or platelet proteins except thrombospondin. Its epitope has been reported to localize in the EGF-like repeats (type 2) in the stalk region of human thrombospondin (disulfide-bonded core remaining after trypsin digestion). Applicant\'s data suggests that Ab-7 may be used to distinguish between lower molecular weight fragments and higher molecular weight fragments (above ˜115 kDa) of thrombospondin or TSP itself, as this antibody recognizes only the high molecular weight fragments or forms. In contrast, no fragments were recognized in normal human or dog plasma with this antibody (See FIG. 8).

All of the antibodies listed above can be purchased from Lab Vision Corporation, Fremont, Calif. currently with a web site at http://www.labvision.com/. See also the published literature such as, for TSP Ab-4, Galvin N J et al. Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy. J Cell Biol. 1987 May; 104(5): 1413-22). It is also understood that alternative antibodies may also be generated against any of the abovementioned epitopes.

5) A purified and/or synthetic thrombospondin fragment, said fragment being at least 6 contiguous amino acyl residues in length, and wherein the fragment does not comprise at least one fibrinogen-binding region selected from the group consisting of (1) a fibrinogen-binding domain within a 210 kDa fragment of TSP, where said 210 kDa fragment is composed of three 70 kDa fragments that contain the region of interchain disulfide bonds, the procollagen homology region, and the type 1 and type 2 repeats, (2) a fibrinogen-binding region in the amino-terminal domain of thrombospondin, (3) a fibrinogen-binding region in an 18 kDa amino-terminal heparin-binding domain of thrombospondin, and (4) a region corresponding to synthetic peptide N12/I encompassing amino acid residues 151-164 (I-151 to P-164) of the N-terminal domain of thrombospondin-1. In a particular embodiment, the fragment does not comprise any of the fibrinogen-binding regions in the group.

6) A purified and/or synthetic thrombospondin fragment, said fragment being at least 6 contiguous amino acyl residues in length, wherein the fragment (1) is recognized by the Ab-7 antibody; and (2) has a high molecular weight preferably above 80 kDa and/or above 115 kDa.

For certain applications of each of the 6 additional aspects, the molecular weight of the thrombospondin fragment exceeds an apparent molecular weight of 80 kDa and/or 115 kDa. For certain applications of each of the 6 additional aspects, the molecular weight of the thrombospondin fragment does not exceed 140 kDa; alternatively does not exceed 65 kDa; or alternatively does not exceed 35 kDa, wherein the size in kDa is the apparent size by gel electrophoresis after disulfide bond reduction. The fragments of the 6 additional aspects of the invention can be used to induce antibodies (and/or other binding molecules) of interest in the diagnostic methods or can be used in diagnostic assays, for example, as calibrators, indicators, and/or competitors. It is understood that a fragment can be derivatized, for example, to incorporate and/or be coupled to a label and/or a carrier.

A fragment that can be as little as 6 amino acyl residues in length is preferably immunoreactive. A typical method for immunizations comprises coupling the peptide to a carrier, such as keyhole limpet hemocyanin or ovalbumin. Said couplings to a carrier are also contemplated in the current invention.

The inclusion of the central protease-resistant core domain in the definition of the fragments follows from considerations discussed elsewhere herein. This domain is considered to comprise locations in the mature, thrombospondin protein selected from the group consisting of: a domain of interchain disulfide bonds (around Cys-252 and Cys-256, preferably residues 241-262); the procollagen homology domain (residues 263-360); the type 1 repeats (residues 361-530); the type 2 repeats (residues 531-673); there is a short segment (residues 674-697) between the type 2 repeat domain and the type 3 repeat domain; and then the type 3 repeats (residues 698-925); see FIG. 1 of this Application for examples of protease-resistant fragments that have been reported after artificial digestions in vitro; Chapter 2, “The primary structure of the thrombospondins” in The Thrombospondin Gene Family by J C Adams, R P Tucker, & J Lawler, Springer-Verlag: New York, 1995, pp. 11-42, particularly p. 12; and Chapter 6, “Mechanistic and functional aspects of the interactions of thrombospondins with cell surfaces,” ibidem, pp. 105-157, particularly p. 115. Interchain disulfide bonds (in the region of residues 241-262) are often preserved in protease-resistant fragments. The term “mature”, as used here to refer to the mature thrombospondin protein sequence, means without the 18- to 22-residue signal peptide sequence, here assumed to be 18 residues, following The Thrombospondin Gene Family by J C Adams et al. 1995; see the full human thrombospondin sequence given below in this text; see also FIG. 1 of this application, and the discussions thereof. Nevertheless, it is understood that GenBank file NM—003246.1, also listed as GI:4507484, currently identifies nucleotide residues “112.204” as encoding the signal peptide, which implies a signal peptide of 31 amino acyl residues).

The identification of these peptides, TEENKE (SEQ ID NO:1), LQDSIRKVTEENKE (SEQ ID NO:3), EGEARE (SEQ ID NO:4), PQMNGKPCEGEARE (SEQ ID NO:5), EDTDLD (SEQ ID NO:6), YAGNGHCGEDTDLD (SEQ ID NO:7), CNSPSPQMNGKPCEGEAR (SEQ ID NO:8), RKVTEENKELANELRRP (SEQ ID NO:9), PQMNGKPCEGEAR (SEQ ID NO:11), CEGEAR (SEQ ID NO:12), and RKVTEENKE (SEQ ID NO:13) was achieved by computerized surveys of thrombospondin, the surveys done by request at commercial sources to identify immunogenic regions (epitopes), but these surveys identified many peptides with immunogenic regions, and so the surveys were followed by selection of relevant peptides and/or epitopes based on knowledge of circulating thrombospondin fragments. Other peptides and/or epitopes listed in this application were similarly identified.

A criterion that a fragment comprises an immunogenic and/or immunoreactive portion from a collagen type V binding domain follows from the published properties (e.g., Galvin N J et al. Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy. J Cell Biol. 1987 May; 104(5):1413-22) of the commercially available TSP Ab-4 antibody used below to detect thrombospondin fragments of interest in the plasma. However, Applicant\'s data suggests that the actual epitopes to which these antibodies bind may be different than those that were asserted in the literature.

The collagen V-binding domain of thrombospondin has been mapped to the amino acid sequence corresponding to the region between valine(333) and lysine(412) (V-333 to K-412, using the single-letter symbols V and K for their respective amino acids), inclusive, of human thrombospondin-1 (Takagi T et al. A single chain 19 kDa fragment from bovine thrombospondin binds to type V collagen and heparin. J Biol Chem 268:15544-15549, 1993; as mentioned above, numbers here refer to the mature thrombospondin protein, that is, without the 18- to 22-residue signal peptide sequence, here assumed to be 18 residues). This region would include a portion of the procollagen homology region of thrombospondin and all or nearly all of the first type 1 repeat of thrombospondin (see Chapter 2, “The primary structure of the thrombospondins” in The Thrombospondin Gene Family by J C Adams, R P Tucker, & J Lawler, Springer-Verlag: New. York, 1995, pp. 11-42, but especially p. 24).

The criterion that the fragment comprises an epitope for binding the commercially available TSP Ab-4 antibody follows from the fact that the TSP Ab-4 antibody was used below to successfully detect thrombospondin fragments of interest in the plasma, including the plasma of cancer patients. Significantly, this TSP Ab-4 antibody is described as binding the collagen type V binding domain of thrombospondin.

For references regarding a fibrinogen-binding region within a 210 kDa fragment of TSP composed of three 70 kDa fragments that contain the region of interchain disulfide bonds, the procollagen homology region, and the type 1 and type 2 repeats, see p. 24 of Adams et al. The Thrombospondin Gene Family; citation 53 therein, which is Lawler J et al. Thrombin and chymotrypsin interactions with thrombospondin. Ann NY Acad. Sci. 1986; 485:273-87; and citations immediately below. Additional references for the fibrinogen-binding regions to be excluded include: for a region in an 18 kDa amino-terminal heparin-binding domain of thrombospondin (so-called TSP18), see Bonnefoy A et al.: A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor. J Biol Chem. 2001 Feb. 23; 276(8):5605-12. For a region corresponding to synthetic peptide N12/I encompassing amino acid residues 151-164 of the N-terminal domain of thrombospondin-1, see Voland C et al.: Platelet-osteosarcoma cell interaction is mediated through a specific fibrinogen-binding sequence located within the N-terminal domain of thrombospondin 1. J Bone Miner Res. 2000 February; 15(2):361-368. Citations for two fibrinogen-binding domains include p. 24 of Adams et al. The Thrombospondin Gene Family (and citations 51-54 therein), and for the role of the type 1 repeats include Panetti T S et al.: Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin. J Biol Chem. 1999 Jan. 1; 274(1):430-7.

Thrombospondin is a glycosylated protein. Therefore, depending on which portion of thrombospondin is considered, the thrombospondin fragments of the invention may be glycosylated or non-glycosylated. Potential sites for N-linked carbohydrate chains include N-230 (in the N-terminal domain), N-342 (in the procollagen homology domain), N-503 (in the type 1 repeat domain), N-690 (in the region between the type 2 and type 3 repeat domains), N-1033 (in the C-terminal domain), and N-1049 (in the C-terminal domain). It is also understood that specific C- and O-linked saccharide attachments occur, particularly in the type 1 repeat domain (see Hofsteenge J, Huwiler K G, Macek B, Hess D, Lawler J, Mosher D F, Peter-Katalinic J: C-mannosylation and O-fucosylation of the thrombospondin type 1 module. J Biol Chem. 2001 Mar. 2; 276(9):6485-6498). It is also understood that β-hydroxylation of thrombospondin can occur (such as at N-592, which is in the type 2 repeat domain; see FIG. 2.2a in Adams J C et al. The Thrombospondin Gene Family, 1995, p. 16), and that any of these modifications can be incorporated, or not, into thrombospondin fragments and/or peptides of the current invention.



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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


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