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(12) United States Patent (10) Patent No.: US 8,535,891 B2 Kentsis Et Al

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(12) United States Patent (10) Patent No.: US 8,535,891 B2 Kentsis Et Al US008535891B2 (12) United States Patent (10) Patent No.: US 8,535,891 B2 Kentsis et al. (45) Date of Patent: Sep. 17, 2013 (54) METHOD OF PREDICTING ACUTE 2009/0175844 A1* 7/2009 Nakamura et al. ................ 435/6 APPENDICITIS 2010.0184049 A1* 7, 2010 Goodison et al. .. ... 435/6 2010/0190662 A1* 7/2010 Sutphen et al. ................. 506,18 (75) Inventors: Alex Kentsis, Brookline, MA (US); FOREIGN PATENT DOCUMENTS Hanno Steen, Cambridge, MA (US); WO 2006/125973 11, 2006 Richard Bachur, North Reading, MA (US) OTHER PUBLICATIONS (73) Assignee: Children's Medical Center Mahmoud et al., (Tikrit Medical Journal 2007: 13(1): 81-83).* Corporation, Boston, MA (US) panel et al., (Journal of Leukocyte Biology, 2002; 72(3), 478 (*) Notice: Subject to any disclaimer, the term of this Murphy et al., (Nature 2008; vol. 1 No. 4, published online May patent is extended or adjusted under 35 2.guci ettal. al., Biochem.51Ocnem. J. (2005) 387, 343-353)).* U.S.C. 154(b) by 0 days. Life Science application, Life Technologies 2008 catalog. Aspenbio Pharma, Inc., 'Appendicitis product pipeline.” http://www. (21) Appl. No.: 13/142,598 aspenbiopharma.com/rdproduct/appendicitis; accessed Apr. 7, 2009. (22) PCT Filed: Dec. 30, 2009 Kentsis et al., Annals of Emergency Medicine, 1-9.e4 (2009). “Dis covery and validation of urine markers of acute pediatric appendicitis (86). PCT No.: PCT/US2O09/0698OO using high-accuracy mass spectrometry.” S371 (c)(1), Grosel-Grenc et al., Scandinavian Journal of Clinical and Labora (2), (4) Date: Oct. 14, 2011 lipopolysaccharide-bindingtory Investigation, 67(2): 197-206protein in(2007). acute appendicitis"Interleukin-6 in chiland dren. (87) PCT Pub. No.: WO2010/0784.11 Lycopoulou et al., Clinical Chemistry and Laboratory Medicine, PCT Pub. Date: Jul. 8, 2010 43(1):49-53 (2005). "Serum amyloid A protein levels as a possible aid in the diagnosis of acute appendicitis in children.” (65) Prior Publication Data Sack et al., BMC Surgery, Biomed Central. 6(1): 15-22 (2006). “Diagnostic value of blood inflammatory markers for detection of US 2012/OO28268 A1 Feb. 2, 2012 acute appendicitis in children.” Related U.S. Application Data * cited by examiner (60) Provisional application No. 61/141.283, filed on Dec. 30, 2008, provisional application No. 61/185,676, Primary Examiner — Jacob Cheu filed on Jun. 10, 2009. Assistant Examiner — Carmencita MBelei 74) Attorney,ey, AgAgent, or Firm — David S. Resnick; Tari W. (51) E's (2006.01) Mills: Nixon Peabody LLP GOIN33/536 (2006.01) GOIN 33/537 (2006.01) (57) ABSTRACT GOIN33/54 (2006.01) Embodiments of the invention provide method and devices GOIN33/543 (2006.01) for predicting the likelihood of acute appendicitis without (52) U.S. Cl. invasive exploratory medical procedures. Several protein USPC ............................................ 435/7.1:436/501 biomarkers: leucine-rich C-2-glycoprotein (LRG); S100-A8 (58) Field of Classification Search (calgranulin); C-1-acid glycoprotein 1 (ORM); plasminogen None (PLG); mannan-binding lectin serine protease 2 (MASP2); See application file for complete search history. zinc-C-2-glycoprotein (AZGP1); Apollipoprotein D (Apol)); and C-1-antichymotrypsin (SERPINA3); are increased in the (56) References Cited urine of patients with appendicitis. The method and devices comprise detecting the levels of these biomarkers and com U.S. PATENT DOCUMENTS paring with reference levels found in healthy individuals. 2009/0018026 A1 1/2009 Kim et al. ......................... 506.7 2009.0035875 A1 2/2009 Jemmerson 2009/0104605 A1 4/2009 Siuzdak et al. ................... 435/6 18 Claims, 22 Drawing Sheets U.S. Patent Sep. 17, 2013 Sheet 1 of 22 US 8,535,891 B2 Figure 1 U.S. Patent Sep. 17, 2013 Sheet 3 of 22 US 8,535,891 B2 Figure 3 3 B Error (ppm) U.S. Patent Sep. 17, 2013 Sheet 4 of 22 US 8,535,891 B2 pe?aluoepý? U.S. Patent Sep. 17, 2013 Sheet 5 of 22 US 8,535,891 B2 3. 3 Sue Ol U.S. Patent Sep. 17, 2013 Sheet 6 of 22 US 8,535,891 B2 Figure 6 -1 O 1 Tissue gene Overexpression (log) U.S. Patent Sep. 17, 2013 Sheet 7 of 22 US 8,535,891 B2 - N & S F &aw R Prospective marker validation six U.S. Patent Sep. 17, 2013 Sheet 8 of 22 US 8,535,891 B2 Figure 8 U.S. Patent Sep. 17, 2013 Sheet 9 of 22 US 8,535,891 B2 Figure 9A XXX S ssaxx sym SS xSws & ... -- S 8. S. m (, SS U.S. Patent Sep. 17, 2013 Sheet 10 of 22 US 8,535,891 B2 Figure 10A U.S. Patent Sep. 17, 2013 Sheet 11 of 22 US 8,535,891 B2 igure 1 OB U.S. Patent Sep. 17, 2013 Sheet 12 of 22 US 8,535,891 B2 Figure 11A (Top View) 2.% %2. Figure 11B (Cross Section View) S T C 103 W 101 U.S. Patent Sep. 17, 2013 Sheet 13 of 22 US 8,535,891 B2 Figure 12A Figure 12C Figure 12B Figure 12D C & SS SS U.S. Patent Sep. 17, 2013 Sheet 14 of 22 US 8,535,891 B2 Figure 13 131 U.S. Patent Sep. 17, 2013 Sheet 15 of 22 US 8,535,891 B2 Figure 14 È? U.S. Patent Sep. 17, 2013 Sheet 16 of 22 US 8,535,891 B2 Figure15A Figure15C Figure15B Figure15D U.S. Patent Sep. 17, 2013 Sheet 17 of 22 US 8,535,891 B2 Figure 16A (Top View) Figure 16B (Cross Section View) S T C 163 W 161 U.S. Patent Sep. 17, 2013 Sheet 18 of 22 US 8,535,891 B2 Figure 17A Figure 17B U.S. Patent Sep. 17, 2013 Sheet 19 of 22 US 8,535,891 B2 Figure 17C Figure 17D S U.S. Patent Sep. 17, 2013 Sheet 20 of 22 US 8,535,891 B2 Figure 17E Figure 17F C T HD -CH U.S. Patent Sep. 17, 2013 Sheet 21 of 22 US 8,535,891 B2 Figure 18 U.S. Patent Sep. 17, 2013 Sheet 22 of 22 US 8,535,891 B2 Figure 19 191 US 8,535,891 B2 1. 2 METHOD OF PREDICTING ACUTE In some embodiments, an appendicitis biomarker is leu APPENDICITIS cine C-2 glycoprotein (LRG). In some embodiments, an appendicitis biomarker is mannan-binding lectin serine pro tease 2 (MASP2). In some embodiments, an appendicitis CROSS REFERENCE TO RELATED biomarker is C.-1.-acid glycoprotein 1 (ORM). In some APPLICATIONS embodiments, an appendicitis biomarker is selected from the groups selected from leucine-rich C-2-glycoprotein (LRG); This application is a 35 U.S. C. S371 National Phase Entry S100-A8 (calgranulin); O.-1.-acid glycoprotein 1 (ORM); Application of International Application No. PCT/US2009/ plasminogen (PLG); mannan-binding lectin serine protease 2 069800 filed Dec. 30, 2009, which designates the United (MASP2); zinc-C-2-glycoprotein (AZGP1); apolipoprotein States, and which claims benefit under 35 U.S. C. S 119(e) of 10 D (Apol)); and C-1-antichymotrypsin (SERPINA3). In some U. S. Provisional Application No. 61/141.283, filed Dec. 30, embodiments, an appendicitis biomarker is selected from at 2008, and U. S. Provisional Application No. 61/185,676, filed least 1, or at least about 2, or at least about 3, or at least about Jun. 10, 2009, the contents of which are incorporated herein 4, or at least about 5, or more than 5 of any and all combina by reference in their entireties. tions of appendicitis biomarkers disclosed in Table 1. 15 One aspect of the present invention relates to a device for SEQUENCE LISTING detecting at least one protein biomarker in a urine sample from a subject to identify if the subject is likely to have acute appendicitis, the device comprising: (a) at least one protein The instant application contains a Sequence Listing which binding agent which specifically binds to at least one biom has been submitted in ASCII format via EFS-Web and is arker protein selected from the group of leucine C-2 glyco hereby incorporated by reference in its entirety. This ASCII protein (LRG), mannan-binding lectin serine protease 2 copy, created on Jun. 28, 2011, is named (MASP2), C.-1.-acid glycoprotein 1 (ORM); and (b) at least 20110629 SequenceListing TextFile 701039 one solid Support for the at least one protein binding-agent in 064443 US.txt and is 244,035 bytes in size. (a), wherein the protein-binding agent is deposited on the Solid Support. In some embodiments, a protein-binding agent BACKGROUND 25 deposited on the solid support specifically binds the leucine C-2 glycoprotein (LRG) of SEQ ID NO: 1. In another Appendicitis is a condition characterized by inflammation embodiment, a protein-binding agent deposited on the Solid of the appendix. All cases require removal of the inflamed Support specifically binds to the polypeptide of C-1-acid gly appendix, either by laparotomy or laparoscopy. Untreated, coprotein 1 (ORM) of SEQ ID NO: 3. In another embodi mortality is high, mainly because of peritonitis and shock. 30 ment, a protein-binding agent deposited on the Solid Support Appendicitis is among many human diseases, for which the specifically binds to the polypeptide of mannan-binding lec diagnosis is complicated by the heterogeneity of its clinical tin serine protease 2 (MASP2) of SEQID NO: 5. presentation. Patients with many other disorders can present In some embodiment, the device is useful for detecting with symptoms similar to those of appendicitis. Examples multiple appendicitis biomarkers, for example where the include the following: pelvic inflammatory disease (PID) or device further comprises at least one additional different pro tubo-Ovarian abscess, Endometriosis, ovarian cyst or torsion, 35 tein-binding agent deposited on the Solid Support, wherein the ureterolithiasis and renal colic, degenerating uterine lei additional protein-binding agent specifically binds to a biom omyomata, diverticulitis, Crohn's disease, colonic carci arker protein selected from the group consisting of leucine noma, rectus sheath hematoma, cholecystitis, bacterial rich C-2-glycoprotein (LRG); S100-A8 (calgranulin); O.-1- enteritis, mesentericadenitis, and omental torsion.
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