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(12) United States Patent (10) Patent No.: US 7,537,914 B1 Roux Et Al

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(12) United States Patent (10) Patent No.: US 7,537,914 B1 Roux Et Al USOO7537914B1 (12) United States Patent (10) Patent No.: US 7,537,914 B1 ROux et al. (45) Date of Patent: May 26, 2009 (54) NUCLEICACID AND ALLERGENIC (58) Field of Classification Search ................ 435/69.1, POLYPEPTIDESENCODED THEREBY IN 435/252.3,320.1; 530/350; 536/23.5 CASHEW NUTS (ANACARDIUM See application file for complete search history. OCCIDENTALE) (56) References Cited (75) Inventors: Kenneth Roux, Tallahassee, FL (US); U.S. PATENT DOCUMENTS Shridhar K. Sathe, Tallahassee, FL (US); Jason M. Robotham, Tallahassee, 6,362,399 B1* 3/2002 Kinney et al. ............... 800/312 FL (US); Suzanne S. Teuber, Davis, CA (US) OTHER PUBLICATIONS Wang etal, J Allergy Clin Immunol 110: 160-6, 2002.* (73) Assignee: Florida State University Research Chica et al. Curr Opin Biotechnol 16(4):378-84. Aug. 2005.* Foundation, Inc., Tallahassee, FL (US) Witkowski et al. Biochemistry 38(36): 11643-50, Sep. 7, 1999.* Stanley et al. Arch Biochem Biophys 342(2): 244-53, Jun. 1997.* (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 * cited by examiner U.S.C. 154(b) by 323 days. Primary Examiner Phuong Huynh (74) Attorney, Agent, or Firm Allen, Dyer, Doppelt, (21) Appl. No.: 10/529,602 Milbrath & Gilchrist, PA. (22) PCT Filed: Nov. 4, 2003 (57) ABSTRACT (86). PCT No.: PCT/USO3F34960 The invention describes an isolated nucleic acid sequence S371 (c)(1), comprising the nucleotide sequence of SEQ ID NO:1 or a (2), (4) Date: Mar. 30, 2005 degenerate variant of SEQ ID NO:1. The nucleic acid sequence encodes an Ig-Ebinding immunogenic polypeptide (87) PCT Pub. No.: WO2004/042026 the amino acid sequence of which comprises at least one sequence selected from SEQ ID NOS:4-25. The invention PCT Pub. Date: May 21, 2004 additionally provides an in vitro diagnostic test for detecting anti-cashew IgE in a patient. The test comprises reacting the Related U.S. Application Data patient's serum with a purified polypeptide the amino acid (60) Provisional application No. 60/423,556, filed on Nov. sequence of which comprises at least one sequence selected 4, 2002. from SEQ ID NOS:3-25; separating the polypeptide from unreacted patient serum; reacting the polypeptide with a (51) Int. Cl. labeled human IgE-reactive agent after separating from unre CI2P 2 1/02 (2006.01) acted patient serum; separating the polypeptide from unre CI2N I/2 (2006.01) acted labeled human IgE-reactive agent; and detecting CI2N I5/03 (2006.01) labeled human IgE-reactive agent bound to the polypeptide C07K I4/45 (2006.01) after separating from unreacted agent to thereby indicate C7H 2L/04 (2006.01) presence in the patient's serum of anti-cashew IgE. (52) U.S. Cl. ................ 435/69.1; 435/252.3:435/320.1; 530/350; 530/370, 536/23.6 9 Claims, 7 Drawing Sheets U.S. Patent May 26, 2009 Sheet 1 of 7 US 7,537,914 B1 U.S. Patent May 26, 2009 Sheet 2 of 7 US 7,537,914 B1 U.S. Patent May 26, 2009 Sheet 3 of 7 US 7,537,914 B1 U.S. Patent May 26, 2009 Sheet 4 of 7 US 7,537,914 B1 F.G. 1 C. Cav Ms Car Cp Cp S 14 4 11 S 14 ASRQEWQQQID 23 Car Ah3,4-Ms Ahy Ahy Cav 17 12-4 6 9 7 105 YIQAPQQGRQQG|QSGRFIQ 121 Mg Cav Rc Qr Si VAh34 Cp-Rc-Ahy 5 21. 7 8 6 7 41 9-10-7 180 GINPKDIVFQQQQQHQSRGRIN 198 Ah3-4 Or 3-4 7 225 EDNRGGIVKVKD 236 Ah3 Vs Ah3.4 Cav-Vs 4. Ms 19 Cpl. 4- 8-7 245 SRSQSERGSESIEEESEDEKRRWGORDN 271 Ahy Si U.S. Patent May 26, 2009 Sheet 5 of 7 US 7,537,914 B1 F.G. 1 D. EBNA segment 30 LDAEPDNR 38 Tryptic fragment, LDALEPDNK cDNAsegment 72 GLLLPQYSNAPQLIYWWQG 91 Tryptic fragment, GLLVPSYNN-PELVYVVQG Tryptic fragment, GLLVPSYNN-PELWYVVQ cDNAsegment 272 GIEET ICTMR 281 N-terminal fragment, GLEETICTMT M U.S. Patent May 26, 2009 Sheet 6 of 7 US 7,537,914 B1 FIG. 2 3.N OP i :- | 1 8 st: s s-r . .sif: 18 - US 7,537,914 B1 1. 2 NUCLECACID AND ALLERGENC 4. Bhalla, P. L., I. Swoboda, and M. B. Singh. 2001. “Reduc POLYPEPTIDESENCODED THEREBY IN tion in Allergenicity of Grass Pollen by Genetic Engineer CASHEW NUTS (ANACARDIUM ing.” Int Arch Allergy Immunol. 124:51-54. OCCIDENTALE) 5. Bredehorst, R and K David. 2001. “What Establishes a Protein As an Allergen?” Journal of Chromatography B RELATED APPLICATION 756:3340. 6. Burks, A W. JMJames, A Hiegel, G Wilson, JG Wheeler, This application claims priority from U.S. provisional S M Jones, and NZuerlein. 1998. Atopic Dermatitis and application Ser. No. 60/423,556, which was filed on Nov. 4, Food Hypersensitivity Reactions.”J Pediatr 132:132-36. 2002, and which is incorporated herein by reference in its 10 7. Ehrlich P. 1989. “On Immunity With Special Reference to entirety. Cell Life.” Cong of Immunol ’89:178-97. 8. Eigenmann, PA, A W Burks, G A Bannon, and H A FIELD OF THE INVENTION Sampson. 1996. “Identification of Unique Peanut and Soy Allergens in Sera Adsorbed With Cross-Reacting Antibod The present invention relates generally to the field of cell 15 ies.” Allergy Clin Immunol 98:969-78. biology and, more particularly, to a nucleic acid sequence and 9. Fernandez, C., A. Fiandor, A. Matinez-Garate, and Q. J. the polypeptides encoded thereby which contain an allergen Martinez. 1995. Allergy to Pistachio: Cross-Reactivity found in cashew nuts. Between Pistachio Nut and Other Anacardiaceae.” Clin. Exp. Allergy 25:1254-59. BACKGROUND OF THE INVENTION 10. Fujino, K, H Funatsuki, M Inada, Y Shimono, and Y Kikuta. 2001. “Expression, Cloning, and Immunological Allergic reactions to cashew nuts (Anacardium Occiden Analysis of Buckwheat (Fagopyrum Esculentum Moench) tale), though relatively infrequent, can be varied and even life Seed Storage Proteins.” J. Agric. Food Chem. 49:1825-29. threatening reactions include contact or systemic dermatitis 11. Garcia, F, I Moneo, B Fernandez, J M Garcia-Menaya, J (to cardol and anacardic acid found in the cashew nut shell 25 Blanco, SJuste, and J Gonzalo. 2000. Allergy to Anacar oil) (Marks et al. 1984), atopic dermatitis, and IgE-mediated diaceae: Description of Cashew and Pistachio Nut Aller systemic allergic reactions. (Burks et al. 1998; Tariq et al. gens.”J Investig Allergol Clin Immunol 10:173-77. 1996; Garcia et al. 2000) Pistachios and mangos are other 12. Heiss, S. V. Mahler, R. Steiner, S. Spitzauer, C. Sch edible members of the Anacardiaceae family and are aller weiger, D. Kraft, and R. Valenta. 1999. “Component-Re genic with pistachio showing extensive in vitro and possible 30 solved Diagnosis (CRD) of Type I Allergy With Recombi clinical cross-reactivity with cashews. (Fernandez, Fiandor, nant Grass and Tree Pollen Allergens by Skin Testing.” The Martinez-Garate, and Martinez, 1995) (Parra et al. 1993) Journal of Investigative Dermatology 1 13:830-837. (Quercia, Rafanelli, Marsigli, Foschi, and Stefanini 1999). 13. Helm, RM, G Cockrell, C Connaughton, H A Sampson, Cashew nuts are widely used in Snack foods and as an ingre GA Bannon, V Beilinson, D Livingstone, NC Nielsen, and dient in a variety of processed foods such as “butters', bakery 35 A W Burks. 2000a. “A Soybean G2 Glycinin Allergen. 1. and confectionery products. Identification and Characterization.” Int Arch Allergy We have recently demonstrated that the major IgE-reactive Immunol. 123:205-12. proteins in extracts of cashew nut are legumin-like proteins 14. Helm, RM, G Cockrell, C Connaughton, H A Sampson, and 2S albumins as assessed by N-terminal and enzymatic GA Bannon, V Beilinson, N C Nielsen, and AW Burks. fragment sequencing of native proteins. (Teuber, Sathe, 40 2000b. “A Soybean G2 Glycinin Allergen. 2. Epitope Map Peterson, and Roux 2002) We have also constructed a cashew ping and Three-Dimensional Modeling.” Int Arch Allergy nut cDNA library and, upon initial screening, cloned and Immunol. 123:213-19. sequenced an allergen in the 7S Superfamily, which includes 15. Herian, A M, S L Taylor, and RK Bush. 1990. “Identifi vicilin-like and sucrose binding proteins. (Wang et al. 2002) cation of Soybean Allergens by Immunoblotting With Sera Here, we present the sequence and characteristics of a second 45 From Soy-Allergic Adults.” IntArch Allergy Appl Immunol cDNA encoding protein, designated Ana o 2, which is a 92: 193-98. member of the legumin (11S globulin) family of seed storage 16. Kleber-Janke, T R Crameri, UAppenzeller, M. Schlaak, proteins. We also describe its reaction with sera from cashew and W M Becker. 1999. “Selective Cloning of Peanut allergic patients and map its linear epitopes. Allergens, Including Profilin and 2S 1 bumins, by Phage 50 Display Technology.” Int Arch. Allergy Immunol. 119:265 REFERENCES CITED 74. 17. Marks, J. G. J., T DeMelfi, M A. McCarthy, E J Witte, N 1. Bannon, G. A. G. Cockrell, C. Connaughton, C. M. West, Castagnoli, W L Epstein, and RC Aber. 1984. "Dermatitis R. Helm, J. S. Stanley, N. King, P. Rabjohn, H. A. Samp 55 From Cashew Nuts. J. An Acad Dernatol 10:627-31. son, and A. W. Burks. 2001. “Engineering, Characteriza 18. Niederberger, V., P. Stubner, S. Spitzauer, D. Kraft, R. tion and in Vitro Efficacy of the Major Peanut Allergens for Valenta, K. Ehrenberger, and F. Horak. 2001. "Skin Test Use in Immunotherapy.” Int Arch Allergy Immunol. 124: Results but Not Serology Reflect Immediate Type Respi 70-72. ratory Sensitivity: A. Study Performed With Recombinant 2. Beardslee, TA, MG Zeece, G Sarath, and J P Markwell.
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