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

Abstract: 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.

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.

SUMMARY

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.

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 I 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.

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).

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 10kDa, 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.

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.

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).

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.

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.

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.

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), YAGNGIICGEDTDLD (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.

Nonglycosylated entities of particular interest are synthetic peptides.

In particular embodiments, the thrombospondin fragments of the invention are derivatized so that they comprise and/or are linked to a detectable label and/or a carrier. In particular embodiments, the label is selected from the group consisting of a radioactive label, a fluorescent label, a chemical label, a colorometric label, an enzymatic label, a non-fluorescent label, a non-radioactive label, a biotin moiety, and an avidin moiety. In particular embodiments, the carrier is selected from the group consisting of a bead, a microsphere, a coded microsphere, a solid matrix, a keyhole limpet hemocyanin, an albumin, linkage to a cross-linking agent, an epitope tag, and an epitope.

It is understood that a synthetic or purified thrombospondin fragment of the invention retains its identity as a fragment of the invention even if it has been derivatized by the addition of additional material, such as a detectable label, or through conjugation to another molecule, or by synthesizing it as part of a chimeric protein, to name just three of many possible examples.

The detection of either thrombospondin fragments or thrombospondin usually requires the use of agents that will bind to them. Such agents may be multi-chain antibodies, single-chain antibodies, proteins that are not antibodies, non-protein molecules, or derivatives or combinations thereof. Polyclonal and monoclonal antibodies are normally immunoglobulins, i.e., multi-chain antibodies. In the case of immunoglobulin-G (IgG), each antibody molecule consists of a pair of heavy chains and a pair of light chains. The multichain antibodies are typically from mammalian or avian sources. Single-chain antibodies and non-antibodies are discussed below.

The term “antibodies” by itself, when not specified as being a single-chain antibody, refers to 4-chain antibodies, those with two heavy and two light polypeptide chains. By way of example, this includes but is not limited to the IgG classes of antibodies, but also other classes, such as ones that occur in higher multimers, such as IgM. IgA and IgY are also contemplated.

The term “protein” is intended to include not only molecules normally referred to as proteins but also those that may be referred to as polypeptides.

Methods of Detecting the Thrombospondin Fragments while Distinguishing, or not Distinguishing, from Thrombospondin Itself

In one such aspect, the invention includes an assay to detect a thrombospondin fragment of the invention wherein the assay distinguishes the thrombospondin fragment from thrombospondin itself. Thrombospondin fragments of particular interest are ones found in humans and are within a range selected from the group consisting of 80 to 140 kDa, 40 to 55 kDa and 20 to 30 kDa, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction. Preferably they are selected from the group consisting of a ˜100 to 130 kDa fragment, ˜85 kDa to 90 kDa fragment, an ˜50 kDa fragment, and an ˜30 kDa fragment. The assay may detect just one such fragment, or a combination of 2 or more.

In a preferred embodiment, a higher molecular weight fragment is detected in a sample, wherein the fragment has a molecular weight higher than ˜115 kDa and may be detected by one of the following antibodies: TSP Ab-2, Ab-4, Ab-5, Ab-7 or Ab-8.

In another aspect, this invention contemplates a method of distinguishing between cancerous and non-cancerous plasma samples. This method utilizes an antibody to detect a high molecular weight thrombospondin fragment or fragments that are greater than ˜115 kDa in cancerous plasma samples that are not detectable in non-cancerous plasma samples. In a preferred embodiment, the TSP Ab-2, Ab-4, Ab-5 or Ab-8 antibody is used for detection of the high molecular weight fragments of thrombospondin. In a more preferred embodiment, the TSP Ab-7 antibody is used to recognize the high molecular weight fragments of thrombospondin. Other binding agents, including but not limited to an aptamer, are also contemplated. Automated, high throughput assays are also contemplated.

In a further embodiment, the invention includes a method of distinguishing between improperly collected plasma samples and properly collected plasma samples. This method includes analyzing various plasma samples with an antibody or other binding agent that recognizes high molecular weight thrombospondin fragments in improperly collected normal plasma samples and not in properly collected normal plasma samples. In a preferred embodiment, the antibody used for detection is TSP Ab-2, Ab-4, Ab-5, Ab-7, or Ab-8. Most preferable of these antibodies is Ab-7. Other binding agents, including but not limited to an aptamer, are also contemplated. Automated, high throughput assays are also contemplated.

In cases where the concentration of higher molecular weight forms of thrombospondin, including thrombospondin itself, is low in a sample (such as in the samples shown in FIGS. 3, 4 and 6), detection of fragments without necessarily excluding thrombospondin is an approach also contemplated by the current invention. Low concentrations of thrombospondin can be achieved in many cases by preventing or reducing platelet activation during sample collection and/or storage (see below for contemplated methods). This aspect of the current invention comprises several advantages over conventional detection methods that have used binding agents against the entire thrombospondin molecule (and these binding agents have been limited to antibodies). Said advantages include but are not limited to the use of binding agents that are directed specifically against the fragments of interest and not portions of the thrombospondin molecule outside of these fragments, the use of relevant peptides and/or thrombospondin fragments to generate said binding agents (such as antibodies), the use of relevant peptides and/or thrombospondin fragments as assay calibrators, and the use of relevant peptides and/or thrombospondin fragments as assay indicators.

Any of several acceptable approaches can be used for the assay of a thrombospondin fragment (or fragments) wherein the assay distinguishes it from thrombospondin, and more than one of these can be used in a given assay. In one approach, the assay comprises a step wherein the fragment is physically separated from the thrombospondin. Generally that approach is combined with a step in which the presence of the fragment or thrombospondin is shown by their reaction with a specific binding agent. Assay methods include but are not limited to those well-known in the art, such as ELISA, radioimmunoassay, Western blotting, immunohistochemistry, immunofluorescence, other immune-based methods, non-immune-based methods, quantitative methods, high throughput methods, automated methods, semi-quantitative methods and qualitative methods. In particular embodiments, the physical separation technique is selected from the group consisting of gel electrophoresis, dialysis, chromatography, size chromatography, affinity chromatography, immunoaffinity chromatography, adsorption, immunoadsorption, isoelectric focusing, mass spectrometry, centrifugation, sedimentation, floatation, precipitation, immunoprecipitation, and gel filtration.

In a second approach, the assay distinguishes the fragment (or fragments) based on one or more epitopes (here “epitope” meaning a target to which a binding agent, i.e., an antibody or a non-antibody, binds) in the fragment that are not present in thrombospondin, including but not limited to an epitope at an end of a fragment and an epitope that is displayed by a fragment but is shielded in thrombospondin.

In a third approach, the assay distinguishes the fragment (or fragments) based on one or more epitopes in thrombospondin that are not present in the fragment. As an illustrative but not restrictive example, an epitope shared by thrombospondin and a thrombospondin fragment is used to obtain a quantitation of a total, thrombospondin plus thrombospondin fragment(s), from which is then subtracted a quantitation of thrombospondin obtained using an epitope present in thrombospondin but not present in a fragment. The difference between the two quantitations is a quantitation of the amount of fragment. As an example, epitopes in thrombospondin but not in at least one fragment from the group of an 80 to 140 kDa, a 40 to 55 kDa, or a 20 to 35 kDa fragment present in plasma can be selected from the group consisting of an epitope from outside the protease-resistant central core domain, an epitope in the N-terminal, domain, an epitope in the N-terminal heparin-binding domain, a heparin-binding sequence in the N-terminal domain, a heparin-binding sequence in the N-terminal domain selected from the group consisting of residues 23-32 (RKGSGRRLVK), residues 23-29 (RKGSGRR), and residues 77-83 (RQMKKTR) of the mature protein (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. 13 & Table 2.1; Chapter 6, “Mechanistic and functional aspects of the interactions of thrombospondins with cell surfaces,” ibidem pp. 105-157, but especially pp. 108 & 114; Lawler J et al. Expression and mutagenesis of thrombospondin. Biochemistry. 1992 Feb. 4; 31(4):1173-80; and Cardin A D & Weintraub H T. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis. 1989 January-February; 9(1):21-32), a heparin-binding sequence in the N-terminal domain selected from the group consisting of residues 22-29 (ARKGSGRR), residues 79-84 (MKKTRG), and residues 178-189 (RLRIAKGGVNDN) of the mature protein (reviewed in the Discussion section of 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), an epitope in the C-terminal domain, an epitope in the C-terminal cell-binding domain, a thrombospondin epitope not found in a plasma fragment, a thrombospondin epitope not found in a plasma fragment of 80 to 140 kDa, a thrombospondin epitope not found in a plasma fragment of 40 to 55 kDa, and a thrombospondin epitope not found in a plasma fragment of 20 to 35 kDa, where all kDa molecular weights are those after reduction. It is understood that the absence of a strong, functional heparin-binding domain from a thrombospondin fragment in plasma will be a factor allowing its accumulation in plasma (many heparin- or heparan-binding proteins are cleared from plasma very quickly; see for example, Wallinder L et al. Rapid removal to the liver of intravenously injected lipoprotein lipase. Biochim Biophys Acta. 1979 Oct. 26; 575(1):166-73).

The epitopes may be divided into three Groups. Group 1: An epitope shared by thrombospondin and a thrombospondin fragment present in plasma is preferably one that is contained within 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 (SEQ ID NO: 10), PQMNGKPCEGEAR (SEQ ID NO:11), CEGEAR (SEQ ID NO:12), RKVTEENKE (SEQ ID NO:13), or a portion at least 3 amino acyl residues in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues) of such an amino acid sequence.

Group 2: An epitope in thrombospondin but not in an 80 to 140 kDa, 40 to 55 kDa, and/or 20 to 35 kDa fragment present in plasma is preferably one contained within an amino acid sequence selected from the group consisting of TERDDD (SEQ ID NO: 24), DFSGTFFINTERDDD (SEQ ID NO: 25), ERKDHS (SEQ ID NO: 26), TRGTLLALERKDHS (SEQ ID NO: 27), CTRGTLLALERKDHS (SEQ ID NO: 28) (which includes an N-terminal Cys added to aid conjugation), DDKFQD (SEQ ID NO: 29), ANLIPPVPDDKFQD (SEQ ID NO: 30), CANLIPPVPDDKFQD (SEQ ID NO: 31) (which includes an N-terminal Cys added to aid conjugation), DCEKME (SEQ ID NO: 32), EDRAQLYIDCEKMEN (SEQ ID NO: 33) (although it is understood that this sequence and its fragments impinge on the sequence of the fibrinogen-binding N12/I peptide), CGTNRIPESGGDNSVFD (SEQ ID NO: 34), NRIPESGGDNSVFD (SEQ ID NO: 35), GWKDFTAYRWRLSHRPKTG (SEQ ID NO: 36), CGWKDFTAYRWRLSHRPKTG (SEQ ID NO: 37) (which includes an N-terminal Cys added to aid conjugation), or a portion at least 3 amino acyl residues in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues) of such an amino acid sequence.

Various modifications, such as a C-terminal Cys, can be added to a peptide of interest to allow easier conjugation to a carrier protein such as KLH, ovalbumin, and others. This is particularly true for the following peptides: RKVTEENKELANELRRP (SEQ ID NO: 9), LQDSIRKVTEENKE (SEQ ID NO: 3); TRGTLLALERKDHS (SEQ ID NO: 27), and ANLIPPVPDDKFQD (SEQ ID NO: 30), and these modifications provide alternative conjugation strategies for NRIPESGGDNSVFD (SEQ ID NO: 35) and others.

In approaches related to the above, the assay can distinguish fragments from each other, based on physical separation methods and/or on shared and/or non-shared binding agent targets. Thus, for example, size-exclusion chromatography and/or SDS-polyacrylamide gel electrophoresis can be used to separate the ˜85 to 90, ˜50, and ˜30 kDa fragments from each other, for separate quantitation (an example of this is shown in FIG. 3, with the quantitation presented in Table 2). Also, for example, an epitope (meaning a binding agent target) in the ˜85 to 140 kDa fragment that is not contained in the ˜50 kDa and/or the ˜30 kDa fragments can be used to assay it separately, and/or can be used to subtract its contribution from a total to obtain results reflective of the smaller fragments.

Group 3: An additional epitope, useful as a binding agent target for distinguishing a fragment from full-length TSP, and/or distinguishing two fragments of different sizes is preferably one contained within an amino acid sequence selected from the group consisting of DDDDNDKIPDDRDNC (SEQ ID NO: 14), DDDDNDKIPDDRDNC[NH2] (SEQ ID NO: 15), DDDDNDK (SEQ ID NO: 16), NLPNSGQEDYDKDG (SEQ ID NO: 17), CNLPNSGQEDYDKDG (SEQ ID NO: 18), EDYDKD (SEQ ID NO: 19), CPYNHNPDQADTDNNGEGD (SEQ ID NO: 20), CRLVPNPDQKDSDGD (SEQ ID NO: 21), DQKDSDGD (SEQ ID NO: 22), CPYVPNANQADHDKDGKGDA (SEQ ID NO: 23), or a portion at least 3 amino acyl residues in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues) of such an amino acid sequence.

It is also understood that some peptides that contain an epitope shared by thrombospondin and a first thrombospondin fragment present in plasma may contain an epitope that is not shared by a second thrombospondin fragment present in plasma. Said peptides are useful in many applications described herein, including but not limited to distinguishing thrombospondin from said second thrombospondin fragment, distinguishing said first from said second thrombospondin fragment, detecting and/or quantitating thrombospondin, detecting and/or quantitating said first thrombospondin fragment, detecting and/or quantitating said second thrombospondin fragment (in a combination assay described elsewhere herein), and producing a binding agent. Said peptides, which form a subset of Group 1, can be selected from the group consisting of 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), PQMNGKPCEGEAR (SEQ ID NO: 11), CEGEAR (SEQ ID NO: 12), or a portion at least 3 amino acyl residues in length (preferably at least 4 amino acyl residues in length, more preferably at least 6 amino acyl residues) of such an amino acid sequence.

It is also understood that the current invention also includes antibody and non-antibody molecules that bind these peptides, other peptides of thrombospondin specified herein, fragments thereof, and peptides that contain fragments thereof; as well as assays using a reagent from this list. It is understood that an antibody or a non-antibody that distinguishes thrombospondin from a fragment, or one fragment from another, can be employed to affinity-purify thrombospondin or a fragment.

In embodiments of particular interest, a sample of material (liquid tissue, solid tissue, urine, perspiration, cerebrospinal fluid, a body fluid, blood or a blood component, or stool; most preferably blood plasma) is taken or gathered from an organism (either a human or a non-human, preferably a mammal or a bird in the case of non-humans) and is subject to the assay. The inventions disclosed herein not only apply to fragments of human thrombospondin, but also to fragments of non-human thrombospondin. For example, there is a need to detect the presence of or monitor the status of disease, such as a cancer, in livestock, racehorses, pets, and other economically and/or emotionally important animals. The current inventions meet these needs.

In one set of embodiments, the assay detects the presence of, or monitors the course of, diseases and conditions that can affect plasma levels of thrombospondin fragments. Such diseases include, but are not limited to, many that in the prior art were assumed to affect plasma levels of thrombospondin: a cancer, renal failure, renal disease, atopic dermatitis, vasculitis, acute vasculitis, renal allograft, allergic asthma, diabetes mellitus, myocardial infarction, liver disease, splenectomy, dermatomyositis, polyarteritis nodosa, systemic lupus erythematosus, lupus erythematosus, Kawasaki syndrome, non-specific vasculitis, juvenile rheumatoid arthritis, rheumatoid arthritis, vasculitis syndrome, Henoch-Schönlein purpura, thrombocytopenic purpura, purpura, an inflammatory condition, a condition associated with clotting, a condition associated with platelet activation, a condition associated with intravascular platelet activation, a condition associated with consumption of platelets, heparin-induced thrombocytopenia, disseminated intravascular coagulation, intravascular coagulation, extravascular coagulation, a condition associated with endothelial activation, a condition associated with production and/or release of thrombospondin and/or a thrombospondin fragment, urticaria, hives, angioedema, a drug reaction, an antibiotic reaction, an aspartame reaction, atopic dermatitis, eczema, hypersensitivity, scleroderma, conditions associated with plugging of vessels, a condition associated with a cryofibrinogen, a condition associated with a cryoglobulin, and a condition associated with an anti-cardiolipin antibody.

In embodiments of particular interest, the assay for thrombospondin fragments is done to detect the presence of, or monitor the status of, a cancer in a human and/or in a non-human animal. In additional embodiments of interest, the assay is done to measure the degree of platelet activation.

In measurements of plasma levels of the fragments, it is preferred that the plasma is obtained by a method that prevents or reduces platelet activation and/or activation of a component of the clotting cascade during sample collection and/or storage; and/or by a method that prevents or reduces cleavage of thrombospondin into fragments (or fragments into smaller fragments) during sample collection and/or storage. Platelet activation and/or activation of a component of the clotting cascade during sample collection and/or storage can result in the release of thrombospondin, but also activation of proteases (including but not limited to a protease of the clotting cascade) that can cleave thrombospondin and some thrombospondin fragments, thereby complicating the assay. To prevent or reduce platelet activation during sample collection and/or storage, the method may be one that does not comprise the use of a tournequet. Also to prevent or reduce platelet activation and/or activation of clotting during sample collection and/or storage, the method may, for example, comprise a step selected from the group consisting of: (1) use of a large-bore needle, (2) discarding of the initial portion of the collected blood, (3) use of a coated needle, (4) use of a coated tubing, (5) storage of sample between −1° C. and 5° C., and (6) separation of plasma within 30 minutes of sample collection. Also to prevent or reduce platelet activation and/or protease activity during sample collection and/or storage, the method may comprise the use of an agent the use of an agent selected from the group consisting of a platelet inhibitor, a protease inhibitor, a serine protease inhibitor, an enzyme inhibitor, an inhibitor of an enzyme that is divalent cation dependent, a heparin, a heparin fragment, a heparan, an anticoagulant, a COX inhibitor, an inhibitor of a cell-adhesion molecule, an inhibitor of a surface receptor, a glycoprotein inhibitor, an inhibitor of a glycoprotein IIb/IIIa receptor, a thrombin inhibitor, an inhibitor of degranulation, a chelator, a citrate compound, theophylline, adenosine, and dipyridamole (Diatube H vacutainers containing citrate, theophylline, adenosine, and dipyridamole are commercially available from Becton Dickinson; see Bergseth G et al. A novel enzyme immunoassay for plasma thrombospondin: comparison with beta-thromboglobulin as platelet activation marker in vitro and in vivo. Thromb. Res. 99:41-50, 2000; such tubes can be referred to as CTAD tubes). Devices that minimize platelet activation and/or protease activity in a sample are also contemplated and include, but are not limited to, a collection tube containing a cocktail of platelet and/or clotting inhibitors, a collection tube containing a protease inhibitor, a collection tube containing an inhibitor of a protease that is or is derived from a blood component, and a device that discards or allows the easy discarding of the initial portion of collected blood. These methods can also be applied to samples of other body fluids.

A related aspect of the invention is a combination diagnostic test (especially for cancer) comprising at least two types of diagnostic tests, one of said tests being the assay for a thrombospondin fragment (or fragments) or a portion (or portions) thereof in plasma, the other assay not being based on a thrombospondin fragment or portion. In one set of embodiments, the test not based on a thrombospondin fragment or portion thereof is selected from the group consisting of an imaging test, a radiographic test, a nuclear medicine test, a magnetic resonance imaging test, a blood test, a biopsy, a genetic test, a guaiac test, a test for fecal occult blood, and a test for fecal blood, a cancer test not based on a thrombospondin fragment or portion thereof, a disease test not based on a thrombospondin fragment or portion thereof, and an endoscopy. In particular embodiments of the foregoing methods, a thrombospondin fragment comprises a detectable label (at least during some part of the method).

Detection can, for example, be part of a screening process. Such a screening could include a comparison against a reference value, involve a comparison against a previous value from the same individual; and/or be done repeatedly and/or periodically (e.g., once a year, once every six months, or once every 2, 3, 4, 5 or 10 years.). It is understood that screening can be performed on humans and/or on non-human animals

The foregoing methods are assays to detect a thrombospondin fragment of the invention wherein the assay distinguishes, or does not distinguish, a thrombospondin fragment from thrombospondin, or one thrombospondin fragment from another thrombospondin fragment. In any case, such fragments can be referred to as “target” fragments for purposes of the assay. In many instances it is desirable to have the method also comprise a calibration step or procedure, in which known amounts of a thrombospondin fragment (such as a peptide) are subjected to the method. Such “calibration” fragments are optionally detectably labeled. It is possible to perform the assays in which the target and calibration fragments comprise different detectable labels (or where one is detectably labeled and the other is not).

It is understood that interference resulting from fibrinogen binding to an N-terminal domain of thrombospondin is unlikely to affect the detection of thrombospondin fragments related to the protease-resistant core domain (which lack the N-terminal domain). Nevertheless, assays of thrombospondin could be affected (thus, avoiding that region of the N-terminus when assaying thrombospondin and/or diluting, removing, inhibiting, and/or otherwise compensating for interfering molecules is contemplated).

Additional potentially interfering substances, inferred from reports that these molecules are present in plasma and that they bind TSP, are plasminogen, histidine rich proteins including histidine-rich glycoprotein, and fibronectin (See, for example, Walz D A et al., Semin Thromb Hemost. 13(3):317-025 (1987); Vanguri V K et al., Biochem J. 2000 Apr. 15; 347(Pt 2):469-73). For binding of histidine-rich glycoprotein, two regions of thrombospondin have been implicated: type 1 repeats (Simantov et al. J Clin Invest. 2001 January, 107(1):45-52) and a TSP heparin binding domain (Vanguri V K et al., 2000). The heparin-binding domain of thrombospondin is expected to be absent from the circulating fragments.

To compensate for interfering substances in assays for thrombospondin fragments, diluting, removing, inhibiting, and/or otherwise compensating for interfering molecules is contemplated. As an illustrative, but not limiting, example, the inclusion of an inhibitor of thrombospondin-fibrinogen interactions is contemplated. Such an inhibitor is selected from the group consisting of 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):361-8), and an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen (see Tuszynski G P et al.: The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction. J Biol. Chem. 1985 Oct. 5; 260(22):12240-5).

Raising conventional antibodies (also referred to herein simply as “antibodies” as opposed to “single chain antibodies”; and an example of a conventional antibody is IgG, which is composed of two heavy chains and two light chains) is merely one of a number of methods that are generally based on the approach of random, semi-random, directed, combinatorial, and/or other means for the generation of large numbers of diverse peptides and/or non-peptides, that is then followed by a selection procedure to identify within this large number those peptides and/or non-peptides that bind to a target and/or an epitope within a target. Selection can then be followed by methods for improving the peptides and/or non-peptides to achieve better affinity and/or specificity. These diverse peptides and/or non-peptides may be conventional multi-chain antibodies (polyclonal or monoclonal), single-chain antibodies, or non-antibodies, including but not limited to peptides, products of phage display, aptamers, DNA, RNA, or modified DNA or RNA. Also contemplated are thrombospondin receptors and/or binding proteins (such as a CSVTCG receptor, a CSVTCG binding molecule, CD36, angiocidin, 26S proteasome non-ATPase regulatory subunit 4, and/or anti-secretory factor).

A well-known procedure for generation of large numbers of diverse peptides is through phage display, which is then followed by selection and can be further refined through other techniques such as molecular evolution (see, for example, Flores-Flores, C. et al, Development of human antibody fragments directed towards synaptic acetylcholinesterase using a semi-synthetic phage display library. J Neural Transm Suppl. 2002; (62):165-179; Qian, M. D, et al, Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a recombinant phage. Human Antibodies. 2002; 11(3):97-105). scFv constructs can be made by linking variable domains of heavy (VH) and light (VL) chains together via a polypeptide linker (for example, see Asvadi P et al. Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD. J Mol Recognit 15:321-330, 2002). Peptides generated then selected (and then possibly improved) via this approach have been used in ELISAs and ELISA-like assays of their targets (e.g., see Schlattner U et al. Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library. Biochim Biophys Acta. 2002 Dec. 12; 1579(2-3):124-32; Oelschlaeger P et al. Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chain antibody fragment and enhanced green fluorescent protein. Anal Biochem. 2002 Oct. 1; 309(1):27; Nathan S et al. Phage display of recombinant antibodies toward Burkholderia pseudomallei exotoxin. J Biochem Mol Biol Biophys. 2002 February; 6(1):45-53; Lu D et al. Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments. J Immunol Methods. 2002 Sep. 15; 267(2):213-26; Zhang W et al. Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies. Lupus. 2002; 11(6):362-9; Reiche N et al. Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens. Infect Immun. 2002 August; 70(8):4158-64; Rau D et al. Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA. J Immunoassay Immunochem. 2002; 23(2):129-43; and Zhou B et al. Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat. Proc Natl Acad Sci USA. 2002 Apr. 16; 99(8):5241-6; Baek H et al., An improved helper phage system for efficient isolation of specific antibody molecules in phage display. Nucleic Acids Res. 2002 Mar. 1; 30(5):e18).

scFv constructs can be based on antibodies, as in most of the references above, on T-cell receptors (e.g., Epel M et al. A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells. Cancer Immunol Immunother. 2002 December; 51(10):565-573), or on other sequences. Different phage coat proteins have been used to display the diverse peptides (see Gao C et al. A method for the generation of combinatorial antibody libraries using pIX phage display. Proc Natl Acad Sci USA. 2002 Oct. 1; 99(20):12612-6). For an example of fusion constructs, see Lu D et al. Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments. J Immunol Methods. 2002 Sep. 15; 267(2):213-26.

For an example of molecular evolution to improve binding affinity, see Rau D et al. Cloning, functional expression and kinetic characterization of pesticide-selective Fab fragment variants derived by molecular evolution of variable antibody genes. Anal Bioanal Chem. 2002 January; 372(2):261-7. Examples of other modifications “to improve affinity or avidity, respectively [include] by mutating crucial residues of complementarity determining regions or by increasing the number of binding sites making dimeric, trimeric or multimeric molecules.” (the quote is from a review article, Pini A & Bracci L, Phage display of antibody fragments. Curr Protein Pept Sci. 2000 September; 1(2):155-169). The initial set of diverse molecules can be enriched by using sequences from animals or humans exposed to or expressing antibodies against the target (see again Zhang W et al. Lupus 2002; and Reiche N et al. Infect Immun 2002).

Single chain antibodies can also be generated by using Escherichia coli (see Sinacola J R & Robinson A S, Rapid folding and polishing of single-chain antibodies from Escherichia coli inclusion bodies, Protein Expr Purif. 2002 November; 26(2):301-308.)

Non-antibodies also include aptamers and non-antibodies that comprise aptamers. Aptamers are DNA or RNA molecules that have been selected (e.g., from random pools) on the basis of their ability to bind to another molecule (discussed for example at the web site of the Ellington lab, in the Institute of Cellular and Molecular Biology, at the University of Texas at Austin, http://aptamer.icmb.utexas.edu/), wherein said molecule can be a nucleic acid, a small organic compound, or a protein, peptide, or modified peptide (such as thrombospondin or a portion thereof.). An aptamer beacon is an example of a non-antibody that comprises an aptamer (See Hamaguchi N et al., Aptamer beacons for the direct detection of proteins. Anal. Biochem. 2001 Jul. 15; 294(2):126-131.)

Angiocidin is a CSVTCG-specific tumor cell adhesion receptor, see patent application WO 0105968, also NCBI protein accession number CAC32386.1 and/or CAC32387.1 (corresponding to nucleotide accession numbers AX077201 and AX077202), the amino acid sequences specified by those two protein accession numbers as of the date of filing of this application being incorporated herein by reference. It is understood that anti-secretory factor cDNA contains essentially identical nucleotide sequence (e.g., accession #U24704, 99% match by BLAST alignment) to that of angiocidin, as does the nucleotide sequence for the proteasome (prosome, macropain) 26S subunit, non-ATPase, 4 (PSMD4; e.g., accession # NM—002810, also 99% match by BLAST). Anti-secretory factor has the same amino acid sequence as angiocidin, except that AX077201 has a 9-bp insert compared to AX077202, which would mean an additional three amino acyl residues in the encoded protein. Thus, the terms herein are used interchangeably. The NCBI summary for NM—002810 is as follows: “The 26S proteasome is a multicatalytic proteinase complex with a highly ordered structure composed of 2 complexes, a 20S core and a 19S regulator. The 20S core is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits. The 19S regulator is composed of a base, which contains 6 ATPase subunits and 2 non-ATPase subunits, and a lid, which contains up to 10 non-ATPase subunits. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway. An essential function of a modified proteasome, the immunoproteasome, is the processing of class I MHC peptides. This gene encodes one of the non-ATPase subunits of the 19S regulator lid. Two alternate transcripts encoding two different isoforms have been described. Pseudogenes have been identified on chromosomes 10 and 21. Transcript Variant: This variant (1) encodes the longer protein (isoform 1).”Other names for the protein from the protein accession file (NP—002801.1) include “proteasome 26S non-ATPase subunit 4 isoform 1; antisecretory factor 1; 26S protease subunit S5a; S5a/antisecretory factor protein; multiubiquitin chain binding protein; 26S proteasome non-ATPase regulatory subunit 4”.

An important aspect of the invention is the discovery of immunoreactive high molecular weight fragments of thrombospondin. These fragments not only useful in the detection of cancer but also in similar methods and applications described for the low molecular weight fragments as well.

In one aspect, the invention includes a purified high molecular weight thrombospondin fragment that has been extracted from a bodily fluid, wherein the purified thrombospondin fragment is immunoreactive with a TSP Ab-7 antibody. In one embodiment, the molecular weight of said fragment is less than 185 kDa. In another embodiment, the molecular weight is from 85 kDa to 185 kDa, or from 115 kDa to 185 kDa.

In another embodiment, the bodily fluid is blood plasma taken from an animal and/or human suspected and/or known to have a cancer. In another embodiment, the purified thrombospondin fragment is immunoreactive with an antibody selected from the group consisting of TSP Ab-2, Ab-4, Ab-5 and Ab-8.

In a further embodiment, the immunoreactive fragment is detected by analysis using a binding agent, said binding agent selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a single-chain antibody, a non-antibody, a protein, a product of phage display, an anti-secretory factor, an aptamer, a DNA, an RNA, a modified DNA, a modified RNA, a carbohydrate, a glycosaminoglycan, a heparin, a glycoprotein, a proteoglycan, and combinations and derivatives thereof. The fragment may comprise a detectable label, wherein said label is either an intrinsic or added moiety, wherein the detectable label is selected from the group consisting of a radioactive label, a fluorescent label, a chemical label, a colorometric label, an enzymatic label, a non-fluorescent label, a non-radioactive label, a biotin label, and an avidin moiety.

In another aspect, the invention includes a method to detect and/or quantify a high molecular weight thrombospondin fragment, said method comprising performing an immunoassay using an antibody selected from the group consisting of TSP Ab-2, Ab-4, Ab-5, Ab-7, Ab-8, and derivatives and combinations thereof. The immunoassay may be a Western Blot, an ELISA, a high throughput immunoassay, an automated immunoassay, a radioimmunoassay, immunohistochemistry, immunofluorescence, other immune-based methods, non-immune-based methods, quantitative methods, high throughput methods, automated methods, and semi-quantitative methods and qualitative methods.

In a preferred embodiment, a recombinant thrombospondin fragment is used as a standard for said immunoassay. The recombinant thrombospondin fragment is stable at 4° C., and does not contain hazardous, infectious agents. The recombinant fragment also does not vary from donor to donor, as it is not collected by a donor but rather produced in an organism such as bacteria.

In another aspect, the invention includes a method of distinguishing between a cancerous sample of bodily fluid and a non-cancerous sample of bodily fluid, said method comprising: 1) performing an assay on said samples using a binding agent; and 2) detecting thrombospondin and/or high molecular weight fragments of thrombospondin.

In another aspect, the invention includes a method of distinguishing between properly and improperly collected plasma samples, said method comprising: 1) performing an assay with a binding agent on said samples, wherein said samples include a normal human serum sample and at least one cancerous plasma sample; and 2) detecting the presence of thrombospondin and/or high molecular weight thrombospondin fragments. In one embodiment, the samples are drawn into a CTAD tube. In another embodiment, the high molecular weight thrombospondin fragments have a molecular weight from 85 kDa to 185 kDa, or from 115 kDa to 185 kDa.

In another aspect, the invention includes a method to detect and/or quantify a high molecular weight thrombospondin fragment or fragment portion thereof. In a preferred embodiment, the method distinguishes the thrombospondin fragment or fragment portion from thrombospondin based on one or more epitopes in the fragment or fragment portion that are not present in thrombospondin. In a further embodiment, the fragment or fragment portion is distinguished based on one or more epitopes in thrombospondin that are not present in the fragments, said, method comprising the steps of: 1) utilizing an epitope shared by thrombospondin and the thrombospondin fragment or thrombospondin portion as a target for a binding molecule, such as an antibody, to obtain a quantitation of a total, thrombospondin plus either the thrombospondin fragment or thrombospondin portion, 2) utilizing an epitope present in thrombospondin but not present in the fragment or thrombospondin portion to obtain a quantitation of thrombospondin only; and 3) utilizing the difference between the quantitations obtained in steps (1) and (2) as a quantitation of the amount of fragment or thrombospondin portion. In one embodiment, the method is applied to a sample of material taken or gathered from an organism. In a preferred embodiment, the organism is a human.

In another preferred embodiment, the method is performed in order to detect the presence of, or monitors the course of, a disease or condition. In another embodiment, the disease or condition is selected from the group consisting of a cancer, renal failure, renal disease, atopic dermatitis, vasculitis, acute vasculitis, renal allograft, asthma, diabetes mellitus, myocardial infarction, liver disease, splenectomy, dermatomyositis, polyarteritis nodosa, systemic lupus erythematosus, lupus erythematosus, Kawasaki syndrome, non-specific vasculitis, juvenile rheumatoid arthritis, rheumatoid arthritis, vasculitis syndrome, Henoch-Schönlein purpura, thrombocytopenic purpura, purpura, an inflammatory condition, a condition associated with clotting, a condition associated with platelet activation, a condition associated with intravascular platelet activation, a condition associated with consumption of platelets, heparin-induced thrombocytopenia, disseminated intravascular coagulation, intravascular coagulation, extravascular coagulation, a condition associated with endothelial activation, a condition associated with production and/or release of thrombospondin and/or a thrombospondin fragment, urticaria, hives, angioedema, a drug reaction, an antibiotic reaction, an aspartame reaction, atopic dermatitis, eczema, hypersensitivity, scleroderma, conditions associated with plugging of vessels, a condition associated with a cryofibrinogen, a condition associated with a cryoglobulin, and a condition associated with an anti-cardiolipin antibody.

In another preferred embodiment, the disease is a cancer selected from the group consisting of an adenoma, adenocarcinoma, carcinoma, lymphoma, leukemia, solid cancer, liquid cancer, metastatic cancer, pre-metastatic cancer, non-metastatic cancer, a cancer with vascular invasion, internal cancer, skin cancer, cancer of the respiratory system, cancer of the circulatory system, cancer of the musculoskeletal system, cancer of a muscle, cancer of a bone, cancer of a joint, cancer of a tendor or ligament, cancer of the digestive system, cancer of the liver or biliary system, cancer of the pancreas, cancer of the head, cancer of the neck, cancer of the endocrine system, cancer of the reproductive system, cancer of the male reproductive system, cancer of the female reproductive system, cancer of the genitourinary system, cancer of a kidney, cancer of the urinary tract, cancer of a sensory system, cancer of the nervous system, cancer of a lymphoid organ, blood cancer, cancer of a gland, cancer of a mammary gland, cancer of a prostate gland, cancer of an endometrial tissue, cancer of a mesodermal tissue, cancer of an ectodermal tissue, cancer of an endodermal tissue, a teratoma, a poorly-differentiated cancer, a well-differentiated cancer, and a moderately differentiated cancer.

In one embodiment, the method is performed to measure the degree of platelet activation. In another embodiment, the method is performed to measure the degree of secretion of thrombospondin and/or a thrombospondin fragment from a tissue selected from the group consisting of a cancer, a neoplasm, an activated endothelium, and a stroma. In another embodiment, the method is performed on plasma that was obtained by a method that prevents or reduces platelet activation and/or protease activity during sample collection and/or storage.

In yet another embodiment, the method further comprises a test not based on a thrombospondin fragment or portion thereof. This test may bee an imaging test, a radiographic test, a nuclear medicine test, a magnetic resonance imaging test, a blood test, a biopsy, a histologic test, a cytologic test, an immunohistologic test, a genetic test, a guaiac test, a test for fecal occult blood, a test for fecal blood, a test for fecal DNA, a test for a fecal cancer marker, a cancer test not based on a thrombospondin fragment or portion thereof, a receptor test, an estrogen receptor test, a disease test not based on a thrombospondin fragment or portion thereof, an endoscopy, an upper gastrointestinal endoscopy, a lower gastrointestinal endoscopy, a colonoscopy, a sigmoidoscopy, a gastroscopy, a laparoscopy, a laparatomy, a lymph node biopsy, a surgery, and a bronchoscopy.

The blood test is selected from the group consisting of a cancer antigen test, a cancer gene test, a cancer DNA test, a cancer mRNA test, a cancer RNA test, a cancer protein test, a cancer glycoprotein test, a cancer carbohydrate test, a cancer lipid test, a prostate specific antigen test, a test of carcinoembryonic antigen, a test of cancer antigen CA-125, a test of alpha-fetoprotein, a test of CA15-3, a test of CA19-9, a test of malignin, a test of anti-malignin antibody, a test of anti-secretory factor, a cancer antigen that contains a carbohydrate epitope, a cancer antigen that contains a protein or polypeptide epitope, a cancer antigen that contains a lipid epitope, a cancer antigen that contains a mixed epitope, CA 27.29, and episialin.

In another embodiment, the thrombospondin fragment or portion thereof comprises a detectable label, wherein the detectable label is a target and/or indicator fragment and wherein a known or unknown amount of an unlabeled or differently labeled fragment is also subjected to the method, said unlabeled or differently labeled fragment being a thrombospondin fragment or portion thereof.

In another aspect, the invention includes a method of producing antibodies against a high molecular weight thrombospondin fragment or a fragment portion thereof, said method comprising administering said fragment, fragment portion or immunogenic portion thereof to an organism capable of producing antibodies.

In yet another aspect, the invention includes a method of producing a peptide or non-peptide binding agent against a high molecular weight thrombospondin fragment or a fragment portion thereof, or epitope therein, said method comprising the steps of 1) a generating step (random, semi-random, directed, combinatorial, and/or other) to generate large numbers (>100) of diverse peptides and/or non-peptides; 2) a selection step to identify within this large number those peptides and/or non-peptides that bind to the thrombospondin fragment, fragment portion, and/or an epitope therein; and 3) optionally an improvement step for improving the peptide or non-peptide binding agent to achieve better affinity and/or specificity.

In another aspect, the invention includes a cell line capable of producing a binding agent produced by the above described methods. In another aspect, the invention includes a kit for the determination of the presence of, and/or the amount of, and/or the concentration of, a thrombospondin fragment in a material taken or gathered from an organism, said kit comprising a high molecular weight thrombospondin fragment or a fragment portion thereof. The invention also contemplates a kit for the determination of the presence of, and/or the amount of, and/or the concentration of, one or more thrombospondin fragments in a material taken or gathered from an organism, said kit comprising a binding agent capable of binding said one or more of said high molecular weight fragments. In a preferred embodiment, the binding agent is produced by the methods described. In another aspect of the invention, the high molecular weight thrombospondin fragment or portion thereof has been derivatized wherein the derivatization is selected from the group consisting of addition of a detectable label, incorporation of a detectable label, conjugation to another molecule, synthesis of the fragment as part of a chimeric protein, linkage to a carrier molecule or particle, linkage to a carrier, linkage to a bead, linkage to a solid matrix, linkage to keyhole limpet hemocyanin, linkage to an albumin, linkage to an ovalbumin, linkage to a cross-linking agent, linkage to an epitope tag, and linkage to an epitope.

The invention also contemplates a kit for the determination of the presence of, and/or the amount of, high molecular weight thrombospondin fragments in a material taken or gathered from an organism, said kit comprising an antibody that will react with said thrombospondin fragments of interest but not with thrombospondin. The method may also comprise the step of binding a binding agent to the thrombospondin fragment or portion thereof.

The invention also contemplates a method to detect the presence and/or clinical course of a neoplastic disease in an individual, wherein the method comprises the steps of: (1) measuring the individual\'s plasma level of a high molecular weight thrombospondin fragment or fragments; (2) utilizing the result of step (1) in a diagnosis as to whether the individual has a neoplastic disease. In one embodiment, the individual referred to therein is a first individual and wherein the method further comprises the steps of: (3) measuring a second individual\'s plasma level of the thrombospondin fragment, said second individual considered to not have neoplastic disease; and (4) utilizing the result of step (3) is the diagnosis of whether the first individual has a neoplastic disease.

In one embodiment, the greater the extent to which the first individual\'s plasma high molecular weight thrombospondin fragment level exceeds the plasma thrombospondin level of the second individual, the more likely that the diagnosis will be that the first individual has a neoplastic disease and/or a neoplastic disease more advanced than that of the second person. In another embodiment, the method further comprises the steps of assaying the individual\'s plasma level of a thrombospondin fragment more than once, and utilizing the change in plasma level from an older to a more recent value to indicate appearance or progression or improvement wherein said appearance or progression is indicated by an increase in the plasma level and said improvement is indicated by a decrease in said plasma level.

In another aspect, the invention includes a method of producing a binding agent against a high molecular weight thrombospondin fragment, said fragment at least 6 amino acyl residues in length, said method comprising binding a phage to said thrombospondin fragment, and wherein the fragment may be further modified to have a modification selected from the group consisting of glycosylation, β-hydroxylation, and addition of groups or moieties to aid conjugation and/or stability. The binding agent may be an antibody induced by either thrombospondin, a modified form of thrombospondin, a fragment of thrombospondin, a fragment of a modified form of thrombospondin, or a modified fragment of thrombospondin.

In yet another aspect, the invention includes a method that distinguishes two thrombospondin fragments from each other, said fragments being a first fragment and a second fragment, respectively, said method comprising the steps of: (1) utilizing an epitope or binding target shared by said first fragment and said second fragment as a target for a binding agent to obtain a quantitation of a total of said first fragment plus said second fragment; (2) utilizing an epitope or binding target present in said first fragment but not present in said second fragment, to obtain a quantitation of said first fragment only; and (3) utilizing the difference between the quantitations in steps (1) and (2) as a quantitation of the amount of said second fragment. In a preferred embodiment, at least one of said first or second fragment is a high molecular weight fragment of thrombospondin.

The invention also contemplates an aptamer which recognizes a high molecular weight thrombospondin fragment, and can distinguish a high molecular weight thrombospondin fragment from a low molecular weight thrombospondin fragment. In a preferred embodiment, the aptamer distinguishes the same thrombospondin fragments as TSP Ab-7.

In another general aspect, the invention includes a method of producing antibodies and/or other binding agents against an above-noted thrombospondin fragment and/or portion thereof, the method comprising administering such a fragment or portion to an organism (especially a mammal or a bird) capable of producing antibodies. These fragments include both the lower molecular weight fragments, as well as the higher molecular weight fragments. The lower molecular weight fragments include the three dominant forms that are ˜30 kDa, ˜50 kDa, and ˜115 kDa. The higher molecular fragments include those that are more than ˜115 kDa but less than 185 kDa. In a preferred embodiment, the higher molecular weight fragments are recognized by TSP Ab-7. It is understood that said antibodies may comprise monoclonal antibodies and/or polyclonal antibodies. For monoclonal antibodies it is understood that cells from the organism are typically used in the production of hybridomas. For production of antibodies, including monoclonal antibodies, it is understood that any of the thrombospondin fragments and/or portions can be conjugated to a carrier molecule, including but not limited to keyhole limpet hemocyanin and bovine serum albumin, to facilitate the antibody response.

A cell and a cell line for producing the aforementioned monoclonal antibodies are aspects of the invention. Examples of such cells include, but are not limited to, hybridomas, transfected cell lines, and infected cells.

Kits related to the above inventions are themselves aspects of the invention. Such kits are, for example, those that facilitate the determination of the presence of, and/or the amount of, and/or the concentration of, a thrombospondin fragment or fragments in a material taken or gathered from an organism. Such kits optionally comprise a thrombospondin fragment or fragments, or a portion or portions thereof, of the invention. Such kits can comprise a binding agent or agents specific for a thrombospondin fragment, or portion thereof, of interest. They optionally comprise binding agents that will react with thrombospondin but not a fragment or fragments, and/or a portion or portions thereof, of interest. They optionally comprise binding agents that distinguish between thrombospondin and a fragment, and/or between one fragment and another. If intended for solid tissue, the kits may comprise a homogenizing means for extracting a fragment into a solution, which optionally may also be provided. Binding agents of the current invention can also be used for other well-known detection methods, including but not limited to immunohistochemistry.

Preferred binding agents are proteins, although non-proteins are also contemplated. Such proteins include both antibodies and nonantibodies.

Optionally, the kits comprise a means for separating or distinguishing a fragment or fragments (or portions thereof) from thrombospondin. The kits can also include a thrombospondin fragment, a peptide derived from such fragment, or a derivatized fragment or peptide, to facilitate detection and calibration.

In one set of embodiments, the kits are adapted for use in an automated assay, such as one using a clinical autoanalyzer.

Particular kit aspects of the invention can also be summarized as follows:

A kit for the determination of the presence of, and/or the amount of, and/or the concentration of, a thrombospondin fragment or fragments in a material taken or gathered from an organism, said kit comprising a thrombospondin fragment or portion thereof.

A kit for the determination of the presence of, and/or the amount of, and/or the concentration of, one or more thrombospondin fragments in a material taken or gathered from an organism, said kit comprising a binding agent capable of binding said one or more fragments.

Particular embodiments are:

Such kits wherein the binding agent comprises a protein.

Such kits wherein said protein comprises an antibody.

Such kits wherein the antibody is a monoclonal antibody or a polyclonal antibody.

Such kits wherein said protein comprises a fragment of an antibody.

Such kits wherein said protein comprises a single-chain antibody.

Such kits wherein said single chain antibody is derived from a phage display library.

Such kits wherein said protein is a non-antibody, the non-antibody being a protein that is neither a multi-chain antibody nor a single-chain antibody.

Such kits wherein said protein non-antibody is selected from the group consisting of a thrombospondin receptor, a thrombospondin receptor that binds within a protease-resistant core region, a thrombospondin receptor that binds a TSP fragment present in the plasma of a cancer patient, a CSVTCG receptor, a CSVTCG binding molecule, a CD36 (which reportedly binds CSVTCG; see Carron J A et al., A CD36-binding peptide from thrombospondin-1 can stimulate resorption by osteoclasts in vitro. Biochem Biophys Res Commun. 2000 Apr. 21; 270(3):1124-7), angiocidin, anti-secretory factor, 26S proteasome non-ATPase regulatory subunit 4, fragments thereof that bind to their respective targets, and combinations, chimeras, and recombinant versions of said receptors and fragments.

Such kits wherein said binding agent comprises a non-protein.

Such kits wherein said binding agent comprises an aptamer.

Such kits wherein said binding agent comprises angiocidin, anti-secretory factor, and/or 26S proteasome non-ATPase regulatory subunit 4.

Other particular kit aspects of the invention can be summarized as follows:

A kit for the determination of the presence of, and/or the amount of, and/or the concentration of, one or more thrombospondin fragments in a material taken or gathered from an organism, said kit comprising a binding agent that will react with thrombospondin but not with a fragment of interest. Particular embodiments are:

Such kits wherein said binding agent comprises a protein;

Such kits wherein said protein comprises an antibody;

Such kits wherein said antibody is a monoclonal antibody or a polyclonal antibody;

Such kits wherein said protein comprises a fragment of an antibody;

Such kits wherein said protein comprises a single-chain antibody;

Such kits wherein said single chain antibody is derived from a phage display library;

Such kits wherein the protein is a non-antibody, the non-antibody being a protein that is neither an antibody nor a single-chain antibody;

Such kits wherein said non-antibody is selected from the group consisting a an integrin, an RGD receptor, an RFYVVMWK receptor, an RFYVVM receptor, an FYVVMWK receptor, an IRVVM receptor, fragments thereof that bind to their respective targets, and combinations, chimeras, and recombinant versions of said receptors, integrins, and fragments; and

Such kits wherein said binding agent comprises an aptamer, meaning a DNA or RNA or related compound, that binds thrombospondin or a thrombospondin fragment.

Such kits wherein said binding agent comprises angiocidin, anti-secretory factor, and/or 26S proteasome non-ATPase regulatory subunit 4.

Several motifs within thrombospondin for binding to many of the receptors referred to above are shown in FIG. 2.2a of Adams, J. C., et al., The thrombospondin Gene Family, Springer Verlag, New York, 1995, p. 16. A CSVTCG receptor, a CSVTCG binding molecule, an angiocidin, an anti-secretory factor, a CD36, and/or fragments and derivatives thereof will be useful for assaying a thrombospondin fragment in a cancer patient.

The invention as it pertains to the detection or monitoring of neoplastic disease can also be summarized as the following:

A method to detect the presence of neoplastic disease in an individual, wherein the method comprises the steps of:

(1) measuring the individual\'s plasma level of a thrombospondin fragment;

(2) utilizing the result of step (1) in a diagnosis as to whether the individual has a neoplastic disease; said fragment being at least 6 contiguous amino acyl residues in length but less than 140 kDa.

A method of distinguishing between a cancerous and non-cancerous plasma sample based upon the presence of high molecular weight fragments of thrombospondin that are detectable with a TSP antibody such as TSP Ab-2, Ab-4, Ab-5, Ab-7 or Ab-8. Other binding agents, including but not limited to an aptamer, are also contemplated. Automated, high through-put assays are also contemplated.

Related is such a method, where the individual referred to therein is a first individual and wherein the method further comprises the steps of:

(3) measuring a second individual\'s plasma level of the thrombospondin fragment, said second individual considered to not have neoplastic disease;

(4) utilizing the result of step (3) is the diagnosis of whether the first individual has a neoplastic disease. For example, such a method wherein the greater the extent to which the first individual\'s plasma thrombospondin fragment level exceeds the plasma thrombospondin level of the second individual, the more likely that the diagnosis will be that the first individual has a neoplastic disease and/or a neoplastic disease more advanced than that of the second person. It is also understood that values from the first individual taken over time can be compared with one another, to assess the likelihood of the appearance of disease and/or progression and/or regression of disease. Particular embodiments are:

Such methods wherein the fragment is selected from the group consisting of an ˜85 to 140 kDa fragment, and ˜50 kDa fragment, and an ˜30 kDa fragment, wherein the size in kDa is that determined by gel electrophoresis after disulfide bond reduction;

Such methods wherein the neoplastic disease is selected from the group consisting of an adenoma, adenocarcinoma, carcinoma, lymphoma, leukemia, and sarcoma;

Such methods wherein the neoplastic disease is an internal cancer; Such methods wherein the neoplastic disease is selected from the group consisting of a cancer of the respiratory system, a cancer of the circulatory system, a cancer of the musculoskeletal system, a cancer of a muscle, a cancer of a bone, a cancer of a joint, a cancer of a tendor or ligament, a cancer of the digestive system, a cancer of the liver or biliary system, a cancer of the pancreas, a cancer of the head, a cancer of the neck, a cancer of the endocrine system, a cancer of the reproductive system, a cancer of the male reproductive system, a cancer of the female reproductive system, a cancer of the genitourinary system, a cancer of a kidney, a cancer of the urinary tract, a skin cancer, a cancer of other sensory organs (such as eye, ear, nose, tongue), a cancer of the nervous system, a cancer of a lymphoid organ, a blood cancer, a cancer of a gland, a cancer of a mammary gland, a cancer of a prostate gland, a cancer of endometrial tissue, a cancer of mesodermal tissue, a cancer of ectodermal tissue, and a teratoma;

Such methods wherein the neoplastic disease is selected from the group consisting of a cancer of solid tissue, a cancer of the blood or the lymphatic system, a non-metastatic cancer, a premetastic cancer, a metastatic cancer, a poorly differentiated cancer, a well-differentiated cancer, and a moderately differentiated cancer.

Such methods wherein the measurement of a plasma thrombospondin fragment level comprises the use of a binding agent, said binding agent being capable of binding said thrombospondin fragment (Such binding agents are discussed above in the context of the kits of the invention); and

In particular embodiments, the thrombospondin fragment is separated from thrombospondin before said fragment is bound to the binding agent.

Such methods wherein said method comprises the use of a binding agent, comprising a binding agent capable of binding thrombospondin but not the thrombospondin fragment. Possible binding agents are discussed above in the context of kits of the invention.

In particular embodiments, the thrombospondin fragment is separated from thrombospondin before said fragment is bound to the binding agent.

Related inventions are:

A method of producing antibodies against a thrombospondin fragment, said method comprising administering said fragment to an organism capable of producing antibodies;

Said method of producing antibodies wherein said fragment is at least 6 amino acyl residues in length but less than 150 kDa or less than 185 kDa. A polyclonal antibody preparation produced by said method;

A monoclonal antibody produced by said method;

A cell line producing said monoclonal antibody; and

A method of producing a binding agent against a thrombospondin fragment, said method comprising the use of phage display.

Said method of producing a binding agent, wherein said method comprises the selection of a thrombospondin-binding or thrombospondin fragment-binding phage from a phage display.

Said method of producing a binding agent, wherein said fragment at least 6 amino acyl residues in length.

A method of distinguishing between properly and improperly collected plasma samples to be used for detecting thrombospondin fragments.

A method of generating other binding agents, including but not limited to an aptamer, against a thrombospondin fragment are also contemplated. Automated, high through-put assay methods are also contemplated.

An additional general aspect of the invention is an assay for the presence of cancer in an organism, said method comprising measuring the extent of platelet activation. An additional general aspect of the invention is an assay for the presence of cancer in an organism, said method comprising measuring the extent of tumor stromal activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic drawing of thrombospondin.

FIG. 2. Results of staining a gel with Coomasie Blue. Lanes, left to right are in the sequence: a lane with the molecular weight standards (Stds), followed by samples A to G.

FIG. 3. Results of Western Blot analysis using TSP Ab-4 antibody and fluorescence detection. Lanes, left to right are in the sequence: a lane with the molecular weight standards (Stds), followed by samples A to G, which correspond to aliquots of the same samples as in FIG. 2.

FIG. 4. Results of Western blot analysis using TSP Ab-4 antibody and colorometric detection. Lanes, left to right are in the sequence: a lane with molecular weight standards, Lane 1 with purified human platelet TSP, Lanes 2-6 with plasma samples A through E (from subjects with cancer), Lanes 7-9 with dilutions of purified human platelet TSP. Samples were processed using urea denaturation before electrophoresis, followed by electrophoresis through a 12% acrylamide gel and enzymatic colorometric detection after blotting.

FIG. 5. Results of Western Blot analysis using TSP Ab-2 antibody and biotinylation/avidin detection (Ab-2 had been biotinylated, and detection was accomplished with avidin-tagged horseradish peroxidase). Lanes, left to right are in the sequence: Lane 1, normal human serum at one-quarter strength (a separate sample from sample F); Lane 2, molecular weight standards; Lane 3, 50 ng of a recombinant human TSP fragment of ˜140 kDa that was engineered to lack the N-terminal heparin-binding domain; Lanes 4-8, plasma samples from human patients with early cancer (samples A-E); Lanes 9-10, normal dog plasma drawn into CTAD tubes; Lane 11, normal human plasma drawn into a CTAD tube (separate sample from sample G); Lane 12, empty.

FIG. 6. Results of Western Blot analysis using TSP Ab-4 antibody and biotinylation/avidin detection (Ab-4 had been biotinylated, and detection was accomplished with avidin-tagged horseradish peroxidase). Lanes, left to right are in the sequence: Lane 1 normal human serum at one-quarter strength; Lane 2, molecular weight standards; Lane 3, 50 ng human platelet TSP; Lanes 4-8, plasma samples from human patients with early cancer; Lanes 9-10, normal dog plasma drawn into CTAD tubes; Lane 11, normal human plasma drawn into a CTAD tube; Lane 12, 50 ng of a recombinant human TSP fragment of ˜140 kDa that was engineered to lack the N-terminal heparin-binding domain.

FIG. 7. Results of Western Blot analysis using TSP Ab-5 antibody and biotinylation/avidin detection (Ab-5 had been biotinylated, and detection was accomplished with avidin-tagged horseradish peroxidase). Lanes, left to right are in a sequence which corresponds to FIG. 6.

FIG. 8. Results of Western Blot analysis using TSP Ab-7 antibody and biotinylation/avidin detection (Ab-7 had been biotinylated, and detection was accomplished with avidin-tagged horseradish peroxidase). Lanes, left to right are in a sequence which corresponds to FIG. 6.

FIG. 9. Results of Western Blot analysis using TSP Ab-8 antibody and biotinylation/avidin detection (the polyclonal Ab-8 had been biotinylated, and detection way accomplished with avidin-tagged horseradish peroxidase). Lanes, left to right are in a sequence which corresponds to FIG. 6.

FIG. 10. Results of Western Blot analysis using TSP Ab-9 antibody and biotinylation/avidin detection (Ab-9 had been biotinylated, and detection was accomplished with avidin-tagged horseradish peroxidase). Lanes, left to right are in a sequence which corresponds to FIG. 6.

FIG. 11. Results of a Control Western Blot analysis using no primary antibody. Lanes, left to right are in a sequence which corresponds to FIG. 6.

FIG. 12. Represents a graphical display of ELISA analysis of TSP and a TSP fragment.

DETAILED DESCRIPTION

The terms “thrombospondin” and “thrombospondin-1” are used interchangeably herein. It is understood that a single “band” on an electrophoresis gel may in fact reflect the presence of a collection of fragments that together form a population that, during gel electrophoresis under reducing conditions, electrophorese at similar rates.

The terms “test” and “assay” are also used interchangeably.

A “purified” fragment is for example (1) one that is found in human plasma and that has been purified (for example has been isolated from gels on which the plasma has been electrophoresed). A purified fragment is not one that is in human plasma, or other part of a human, and that has not undergone at least some degree of purification.

A “synthesized fragment” is, for example, one that has been synthesized in a laboratory (e.g., by recombinant DNA technology or by chemical synthesis) so as to have the primary structure of such a fragment or a portion thereof.

The amino acid sequence of human thrombospondin-1 from GenBank is:

ACCESSION NM 003246 (protein id = NP_003237.1) VERSION NM_003246.1 GI: 4507484 (SEQ ID NO: 38) MGLAWGLGVLFLMHVCGTNRIPESGGDNSVFDIFELTGAARKGSGRRLVK GPDPSSPAFRIEDANLIPPVPDDKFQDLVDAVRAEKGFLLLASLRQMKKT RGTLLALERKDHSGQVFSVVSNGKAGTLDLSLTVQGKQHVVSVEEALLAT GQWKSITLFVQEDRAQLYIDCEKMENAELDVPIQSVFTRDLASIARLRIA

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