US009075045B2

(12) United States Patent (10) Patent No.: US 9,075,045 B2 Zacharias et al. (45) Date of Patent: Jul. 7, 2015

(54) CELL-BASED DETECTION OF APF 4,770,853. A 9, 1988 Bernstein THROUGHTS INTERACTION WITH CKAP4 4.786,589 A 1 1/1988 Rounds

FOR DIAGNOSS OF INTERSTITIAL $723.W . . . A '98. Shoppet al. CYSTITIS 5.530,101 A 6/1996 Queen et al. 5,625,048 A 4/1997 Tsien et al. (71) Applicants: THE COMMONWEALTH 5,656,448 A 8/1997 Kang et al. MEDICAL COLLEGE, Scranton, PA 5,661,035 A 8, 1997 Tsien et al. (US); UNIVERSITY OF FLORIDA 38. A 19, Estal RESEARCH FOUNDATION, INC., 598.1200 A 11/1999 Tsien et al. Gainesville, FL (US) 6,143,502. A 1 1/2000 Grentzmann et al. 7,005,511 B2 2/2006 Tsien et al. (72) Inventors: David Alan Zacharias, St. Augustine, 2.99. R 299; (all st al Ek S. Sonia Lobo Planey, Scranton, 7,250,298k - B2 7/2007 GlicketSee al.a 7,297,529 B2 11/2007 Polito et al. 7,329,735 B2 2/2008 Tsien et al. (73) Assignees: The Commonwealth Medical College, 7,344,893 B2 3/2008 Kirkegaard et al. Scranton, PA (US); University of 7,393,923 B2 7/2008 Tsien et al. Florida Research Foundation, Inc., 2010.0055113 A1* 3/2010 Keay et al...... 424,172.1 Gainesville, FL (US) 2011/0244493 A1 10, 2011 Zacharias et al. (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 WO 2008O14484 A1 1, 2008 U.S.C. 154(b) by 0 days. WO 2008144485 A2 11/2008

(22) Filed: Jun. 6, 2013 Conrads et al. (CKAP4/p63 Is a Receptor for the Frizzled-8 O O related Antiproliferative Factor from Interstitial Cystitis Patients, The (65) Prior Publication Data Journal of Biological Chemistry 281 (49): 37836-37843 (Dec. 8, US 2014/O193835A1 Jul. 10, 2014 2006)).* International Search Report dated Apr. 12, 2010. Related U.S. Application Data Genbank Accession No. NM006825, Aug. 25, 1999. - - - Genbank Accession No. BC082972, Sep. 28, 2004. (62) Division of application No. 13/133,244, filed as Genbank Accession No. BC094824, May 9, 2005. application No. PCT/US2009/067487 on Dec. 10, Planey, Palmitoylation of Cytoskeleton Associated Protein 4 by 2009, now Pat. No. 8,962,341. DHHC2 Regulates antiproliferative Factor-mediated Signaling, (60) Provisional application No. 61/122,157, filed on Dec. Molecular biology of the Cell, vol. 20, 1454-1463, Mar. 1, 2009. 12, 2008. Griesbeck, et al. Reducing the Environmental Sensitivity of Yellow fluorescent Protein, The Journal of Biological Chemistry, vol. 276, (51) Int. Cl. Issue of Aug. 3, pp. 29188-29194, 2001. C07K 4/705 (2006.01) (Continued) GOIN33/50 (2006.01) C07K 9/00 (2006.01) CI2O I/68 (2006.01) Primary Examiner — Gail R Gabel (52) U.S.GOIN33/68 Cl. (2006.01) (74) Attorney,ey, Agent, or Firm — C aesar Rivise,R1V1Se, PC CPC ...... G0IN33/5091 (2013.01); C07K9/005 (2013.01); C07K 14/705 (2013.01): CI2O (57) ABSTRACT I/6897 (2013.01); G0IN33/6893 (2013.01); G0IN 2333/4728 (2013.01); G0IN 2800/34 An assay system designed to detect a protein biomarker in (2013.01) urine that is diagnostic for interstitial cystitis (IC). The pres (58) Field of Classification Search ence of a 9 amino acid glycopeptide, antiproliferative factor USPC ...... 435/7.1, 7.21, 7.5, 287.2, 287.7, 287.9, (APF), in urine is unique to patients with IC. Urine samples 435/288.3, 288.7, 326,334,371; 436/513, from patients who exhibit symptoms consistent with IC are 436/514, 518, 524,528,548, 44, 46, 161, added to the assay system. Binding of APF to the cytoskeletal 436/169,811; 422/402, 409, 82.09 associated protein 4 (CKAP4) is positive for the presence of See application file for complete search history. APF in urine and diagnostic for IC. The diagnostic system is (56) References Cited a significant and Surprising advance in diagnosis of IC and has commercial applications relevant to women and men who U.S. PATENT DOCUMENTS suffer from symptoms consistent with IC. 4,632,901 A 12/1986 Valkirs et al. 4,683, 195 A 7, 1987 Mullis et al. 10 Claims, 28 Drawing Sheets US 9,075,045 B2 Page 2

(56) References Cited Keay, et al (2003) Changes in human bladder epithelial cell expression associated with interstitial cystitis or antiproliferative fac OTHER PUBLICATIONS tor treatment. Physiol Genomics 14, 107-115. Keay, etal (2006) The frizzled 8-related antiproliferative factor from Campbell, etal. A monomeric red fluorescent protein, PNAS, Jun. 11, IC patents inhibits bladder and kidney carcinoma cell proliferation in 2002, vol. 99, No. 12, 7877-7882. Shaner, et al. Improved monomeric red, orange and yellow fluores vitro. European Journal of Cancer Supplements 4, 87-88. cent derived from Discosoma sp. red fluorescent protein, Keay, etal (2003) Antiproliferative factor, heparin-binding epidermal Nature BioTechnology, vol. 22 No. 12, Dec. 2004. growth factor-like growth factor, and epidermal growth factor in men Zhang, et al. (2008) Identificaiton of CKAP4/p63 as a Major Sub with interstitial cystitis versus chronic pelvic pain syndrome. Urol strate of the Palmitoyl Acyltransferase DHHC2, a Putative Tumor ogy 63, 22-26. Suppressor, Using a Novel Proteomics Method. Moll Cell Proteom Keay, et al. (1996) Decreased 3H-thymidine incorporation by human ics 7, 1378-1388. bladder epithelial cells following exposure to urine from interstitial Yanai, etal (2006) Palmitoylation of huntingtin by HIP14 is essential cystitis patients. J Urol 156, 2073-2078. for its trafficking and function. Nat Neurosci 9, 824-831. Zacharias, et al (2013) Antiproliferative Factor-Induced Changes in Keay, et al. An antiproliferative factor from interstitial cystitis Phosphorylation and Palmitoylation of cytoskeleton-Associated pro patients is a frizzled 8 protein related sialoglycopeptide. proc Natl tein-4 Regulate Its Nuclear Translocation and DNA Binding, Interna Acad Sci USA 101, 11803-11808, Aug. 10, 2004. tional Journal of Cell Biology, vol. 2012, Article id 1509, 13 pages. Kim et al. p53 mediates interstitital cystitis antiproliferative factor Chen-Ou-Zhang, et al APF, HB-EGF, and EGF biomarkers in (APF)-induced growth inhibition of human urothelial cells FEBS patients with ulcerative vs. non-ulcerative interstitial cystittis, BMC Let 581, 3795-3799, Aug. 7, 2007. Urology 2005. Klopfenstein, etal (1998) A novel direct interaction of endoplasmic Matika, et al. Antiproliferative factor regulates connective tissue reticulum with . EMBO J 17, 6168-6177. growth factor (CTGF/CCN2 expression in T24 bladder carcinoma Klopfenstien, et al. Subdomain-specific localization of CLIMP-63 cells, Molecular Biology of the Cell, vol. 23, May 15, 2012. (p63) in the is mediated by its luminal alpha Zacharias, et al. Partitioning of Lipid-Modified Monomeric GFPs helical segment. J Cell Biol 153, 1287-1300, Jun. 11, 2001. into membrance Microdomains of Live Cells, Science, vol. 296, May Baird, et al. Biochemistry, mutagenesis, and oligomerization of 3, 2002. DsRed, a red fluorescent protein from coral, PNAS, Oct. 24, 2000, Bano, et al. (1998) Pseudo-enzymatic S-acylation of a myristoylated vol.97, No. 22, 11984-11989. yes protein tyrosine kinase peptide in vitro may reflect non-enzy Lobo, et al Identificatio of a Ras palmitoyltransferase in Sac matic S-acylation in vivo. Biochem J330 (Pt2), 723-731. BiZZoZero, et al (1987) Autoaclation of myelin proteolipid protein charomyces cerevisiae. J Biol Chem 277, 41268-41273, Oct. 25, withacyl coenzyme A.J. Biol Chem 262, 13550-13557. 2002. Bova, et al. (1993) Homozygous deletion and frequentallelic loss of Moran et al (2001) Thiols in cellular redox signalling and control. 8p22 loci in human prostate cancer. Cancer Res 53, Curr Med Chem 8, 763-772. 3869-3873. Mukai. etal (2004) Evidence that the gene encoding ZDHHC8 con Conrads, et al. (2006) CKAP4/p63 is a receptor for the frizzled-8 tributes to the risk of schizophrenia. Nat Genet 36, 725-731. protein-related antiproliferative factor from interstitital cystitis Ohata, et al. (1993) Deletion mapping of the short arm of chromo patients. J. Biol Chem 281, 37836-37843. Some 8 in non-Small cell lung carcinoma. Chromosome Can Emi, et al. (1993) Allelic loss at chromoscome band 8p21.3-p22 is cer 7, 85-88. associated with progression of hepatocellular carcinoma. Genes Wedegaertner, et al. Activation and depalmitoylation of Gs alphs. Cancer 7, 152-158. Cell 77, 1063-1070, Jul. 1, 1994. Fujiwara, et al., (1993) Evidence for the presence of two tumor Schweizer, et al. Retention of p63 in an ER-Golgi intermediate com Suppressor genes on chromosome 8p for colorectal carcinoma. Can partment depends on the presence of all three of its domains and on its cer Res 53, 1172-1174. ability to form oligomers. J Cell Biol 126, 25-39, Jul. 1, 1994. Gupta, et al., (2006) Identification and characterization of p63 Schweizer, et al. (1993) A reversibly palmitoylated resident proetin (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein Abinding pro (p63) of an ER-Golgi intermediate compartment is related to a cir tein, on type II pneumocytes. Am J Physiol Lung Cell Mol Physiol culatory shock resuscitation protein. J Cell Sci 104 (Pt3), 6685-694. 291, L436-446. Smotrys, et al. (2004) Palmitoylation of intracellular signaling pro Ichii, et al. (1993) Detailed analysis of genetic alterationsin teins, regulation and function, Annu Rev Biochem 73, 559-587. colorectal tumors from patients with and without familial Schweizer, etal (1995) Reassessment of the subcellular localization adenomatous polyposis (FAP). Oncogene8, 2399-2405. of p83.J Cell Sci 108 (Pt. 6), 2477-2485. Jones, et al. (1997) Ligand-gated ion channel Subunit partnerships: Sharma, et al. (2008) DHHC2 Affects Palmitoylation, Stability, and GABAA receptor alphaG subunit gene inactivation inhibits delta Functions of Tetraspanins CD9 and CD151. Mol Biol Cell. subunit expression. J. Neurosci 17, 1350-1362. Baird, et al. Circular permutation and receptor insertion within green Keay, et al., Bladder epithelial cells from patients with interstitial fluorescent proteins, Pro. natl. Acad, Sci., USA. vol. 96, pp. 11241 cystitis produce an inhibitor of heparin-binding epidermal growth 11246, Sep. 1999. factor-like growth factor production. U Urol 164, 2112-21 18, Dec. 2000. * cited by examiner U.S. Patent Jul. 7, 2015 Sheet 1 of 28 US 9,075,045 B2

Figure 1. CKAP4:YFP Construct CKAP4:YFP s wo ony pCMV) CKAP4 YFP SV40 (Ahp U.S. Patent Jul. 7, 2015 Sheet 2 of 28 US 9,075,045 B2

Figure 2. Transiently expressed CKAP4:YFP translocates to the nucleus after application of synthetic APF.

CKAP4:YFP Transient Expression in HeLa Cells Prior to Applying APF

CKAP4:YFP Transient Expression in HeLa Cells After Applying APF ph YFP overlay U.S. Patent Jul. 7, 2015 Sheet 3 of 28 US 9,075,045 B2

Figure 3. Localization of endogenous CKAP4 in the absence (A) or presence (B) of APF (US Provisional Patent #P03444US1, Figure 7B; International Provisional Patentif PO3444WOO, Figure 7B).

Untreated APF

s U.S. Patent Jul. 7, 2015 Sheet 4 of 28 US 9,075,045 B2

Figure 4. CKAP4 abundance is increased in nuclear fractions from APF treated Hella Celis. In Hella Cells treated with APF, we observed a 5-fold increase in the abundance of CKAP4 in the nucleus versus untreated cells as determined by Western blot.

Untreated +APF N E. CKAP4

B-tubulin fibrillarin

Nuclear/Cytosolic Ratio 7 6 3 5 4 S 3 2 1 O APF-treated Control U.S. Patent Jul. 7, 2015 Sheet 5 of 28 US 9,075,045 B2

Figure 5. Schematic diagram of the APF diagnostic assay

CKAP4:YFP reporter expressed on plasma Cell images embrane and perinuclear membranes CKAP4:YFP present in the nucleus

The presence of APF in the patient urine sample is reported based on the DRAG5- overlap of CKAP4:YFP stained fluorescence and DRAQ5 Waiti hourther nucleus fluorescence in the nucleus stain ces Patient urine is applied to the with DRAC5 CKAP4:YFP reporter cells take two images 1st of yellow fluorescence; 2nd of red fluorescence CKAP4:YFP absent from nucleus

If there is no APF in the patient urine sample CKAP4:YFP fluorescence does not appear in the DRACR5 nucleus and there is no stained overlap of the Liceus fluorescence with DRACR5 U.S. Patent Jul. 7, 2015 Sheet 6 of 28 US 9,075,045 B2

Figure 6

6A

.

S. a2 s

t

is U.S. Patent Jul. 7, 2015 Sheet 7 of 28 US 9,075,045 B2

Figure 7

siRNA 7A

age f S

e.

CKAP4 WT CKAP4 C10OS 7B U.S. Patent Jul. 7, 2015 Sheet 8 of 28 US 9,075,045 B2

Figure 8

SiRNA

SiRNA + APF

APF

Untreated Control

CKAP4 U.S. Patent Jul. 7, 2015 Sheet 9 of 28 US 9,075,045 B2

Figure 9

9A He is e

is's 50 e s i Se: is ospri ags 'i t APFP

9B

so

E

it-4 r -2 4-1. The APF is U.S. Patent Jul. 7, 2015 Sheet 10 of 28 US 9.075,045 B2

Figure 10 1.4 ZO-l 2

10A 0.8 ZO-1 mRNA Levels (AU) 0.6 :

0.4 0.2 O troom...wawi growollabilitantosaarivo No TX APF APF+Nonsense APFi siRNA

Vimentin 10 1 OB 1.4 1.2 Vimentin mRNA Levels (AU) 0.8 0.6 . 0.4 0.2

APF+Nonsense APF-siRNA

E-Cadherin 100000

10000 ------

1000 E-Cadherin mRNA Levels (AU) 100

Bess:ce: i.e.:ya: No TX APF APF-I-Nonsense APF siRNA U.S. Patent Jul. 7, 2015 Sheet 11 of 28 US 9,075,045 B2

Figure 11

Wief U.S. Patent Jul. 7, 2015 Sheet 12 of 28 US 9,075,045 B2

Figure 12

12A g 8

fieri

12B U.S. Patent Jul. 7, 2015 Sheet 13 of 28 US 9,075,045 B2

Figure 13

fira hairs) U.S. Patent Jul. 7, 2015 Sheet 14 of 28 US 9,075,045 B2

Figure 14

2

cytoplasm x:X

U.S. Patent Jul. 7, 2015 Sheet 16 of 28 US 9.075,045 B2

Figure 15 (B) (continued) ENSTOOOOO3O8387 GCAAGGGCGGCCACCGCGGCGGCGGCGGCGGCGGCggcaagttccticcitcctoctoctocg ENST00000378026 ENSTOOOOO25852 GCAAGGGCGGCCACCGCGGCGGCGGCGGCGGCGGC------ENSTOOOOO3O8387 cctocgcc.gc.cgctg.ccg.ccgcc.gc.cgcctcgtCctcgg.cgtcctgctcgc.gcaggCTCG ENSTOOOOO37 8026 ENST0000025851.2 ENSTOOOOO3O8387 GCAGGGCGCTCAACTTTCTCTTCTACCTCGCCCTGGTGGCGGCGGCCGCTTTCTCGGGCT ENSTOOOOO378026 ----GGCGCTCAACTTTCTCTTCTACCTCGCCCT----GCGGCGGCCGCTTTCTCGGGCT ENSTOOOOO25851.2 GCAGGGCGCTCAACTTTCTCTTCTACCTCGCCCTGGTGGCGGCGGCCGCTTTCTCGGGCT ENSTOOOOO3O8387 GGTGCGTCCACCACGTCCTGGAGGAGGCCAGCAGGTCCGGCGCAGCCACCAGGACTTC ENSTOOOOO378026 GGTGCGTCCACCACGTCCTGGAGGAGGTCCAGCAGGTCCGGCGCAGCCACCAGGACTTCT ENST0000025852 GGGCGTCCACCACGTCCTGGAGGAGGTCCAGCAGGTCCGGCGCAGCCACCAGGACTTCT ENSTOOOOO3O8387 CCCGGCAGAGGGAGGAGCTGGGCCAGGGCTTGCAGGGCGTCGAGCAGAAGGTGCAGTCTT ENSTOOOOO378026 CCCGGCAGAGGGAGGAGCTGGGCCAGGGCTTGCAGGGCGTCGAGCAGAAGGTGCAGTCTT ENSTOOOOO258512 CCCGGCAGAGGGAGGAGCTGGGCCAGGGCTTGCAGGGCGTCGAGCAGAAGGTGCAGTCTT ENSTOOOOO3O8387 TGCAAGCCACATTTGGAACTTTTGAGTCCATCTTGAGAAGCTCCCAACATAAACAAGACC ENSTOOOOO378026 TGCAAGCCACATTTGGAACTTTTGAGTCCAFCTTGAGAAGCTCCCAACATAAACAAGACC ENSTOOOOO25851.2 TGCAAGCCACATTGGAACTTTTGAGTCCATCTTGAGAAGCTCCCAACATAAACAAGACC ENSTOOOOO3O8387 CACAGAGAAAGCTGTGAAGCAAGGGGAGAGTGAGGTCAGCCGGATCAGCGAAGFGCTGC ENSTOOOOO378026 TCACAGAGAAAGCTGTGAAGCAAGGGGAGAGTGAGGTCAGCCGGATCAGCGAAGTGCTGC ENSTOOOOO25851.2 TCACAGAGAAAGCTGTGAAGCAAGGGGAGAGTGAGGTCAGCCGGATCAGCGAAGTGCTGC ENSTOOOOO3O8387 AGAAACTCCAGAATGAGATTCTCAAAGACCTCTCGGATGGGATCCATGTGGTGAAGGACG ENST00000378026 AGAAACTCCAGAATGAGATTCTCAAAGACCTCTCGGATGGGATCCATGTGGTGAAGGACG ENSTOOOOO25852 AGAAACTCCAGAATGAGACCAAAGACCTCTCGGATGGGATCCAGTGGTGAAGGACG ENSOOOOO3O8387 CCCGGGAGCGGGACTTCACGTCCCTGGAGAACACGGTGGAGGAGCGGCTGACGGAGCTCA ENSTOOOOO37 8026 CCCGGGAGCGGGACTTCACGTCCCTGGAGAACACGGGGAGGAGCGGCTGACGGAGCTCA ENSOOOOO258512 CCCGGGAGCGGGACTTCACGTCCCTGGAGAACACGGTGGAGGAGCGGCTGACGGAGCTCA ENSTOOOOO3O8387 CCAAATCCATCAACGACAACATCGCCATCTTCACAGAAGTCCAGAAGAGGAGCCAGAAGG ENSTOOOOO378026 CCAAATCCATCAACGACAACATCGCCATCTTCACAGAAGTCCAGAAGAGGAGCCAGAAGG ENST00000258,512 CCAAATCCATCAACGACAACATCGCCATCTTCACAGAAGTCCAGAAGAGGAGCCAGAAGG ENST00000308387 AGATCAATGACAFGAAGGCAAAGGTGCCTCCCTGGAAGAATCTGAGGGGAACAAGCAGG ENSTOOOOO378026 AGATCAATGACATGAAGGCAAAGGTTGCCTCCCTGGAAGAATCGAGGGGAACAAGCAGG ENSTOOOOO25851.2 AGATCAATGACATGAAGGCAAAGGTTGCCTCCCTGGAAGAATCGAGGGGAACAAGCAGG ENSTOOOOO3O8387 ATTTGAAAGCCTTAAAGGAAGCTGGAAGGAGATACAGACCTCAGCCAAGTCCAGAGAGT ENSTOOOOO378026 ATTTGAAAGCCTTAAAGGAAGCTGTGAAGGAGATACAGACCTCAGCCAAGTCCAGAGAGT ENSTOOOOO25852 ATTTGAAAGCCTTAAAGGAAGCTGTGAAGGAGATACAGACCTCAGCCAAGCCAGAGAGT ENSTOOOOO3O8387 GGGACATGGAGGCCCTGAGAAGTACCCTTCAGACTATGGAGTCTGACATCTACACCGAGG ENST000003180.26 GGGACATGGAGGCCCTGAGAAG ACCCTTCAGACTATGGAGTCTGACATCTACACCGAGG ENSTOOOOO25851.2 GGGACATGGAGGCCCTGAGAAGTACCCTTCAGACTATGGAGTCTGACATCTACACCGAGG ENSTOOOOO3O8387 TCCGCGAGCTGGTGAGCCTCAAGCAGGAGCAGCAGGCTTTCAAGGAGGCGGCCGACACGG ENST00000378026 TCCGCGAGCTGGTGAGCCTCAAGCAGGAGCAGCAGGCTTTCAAGGAGGCGGCCGACACGG ENST0000025851.2 TCCGCGAGCTGGGAGCCTCAAGCAGGAGCAGCAGGCTTTCAAGGAGGCGGCCGACACGG ENSTOOOOO3O8387 AGCGGCTCGCCCTGCAGGCCCTCACGGAGAAGCTTCTCAGGTCTGAGGAGTCCGTCTCCC ENSTOOOOO378026 AGCGGCTCGCCCTGCAGGCCCTCACGGAGAAGCTTCTCAGGTCTGAGGAGTCCGTCTCCC ENSTOOOOO25851.2 AGCGGCTCGCCCTGCAGGCCCTCACGGAGAAGCTTCTCAGGTCTGAGGAGTCCGTCTCCC ENSTOOOOO3O8387 GCCTCCCGGAGGAGATCCGGAGACTGGAGGAAGAGCTCCGCCAGCTGAAGTCCGATTCCC ENSTOOOOO378026 GCCTCCCGGAGGAGATCCGGAGACTGGAGGAAGAGCTCCGCCAGCTGAAGTCCGATTCCC ENST0000025851.2 GCCCCCGGAGGAGATCCGGAGACTGGAGGAAGAGCTCCGCCAGCTGAAGTCCGATTCCC ENSTOOOOO3O8387 ACGGGCCGAAGGAGGACGGAGGCTTCAGACACTCGGAAGCCTTTGAGGCACCCAGCAAA ENSTOOOOO378O26 ACGGGCCGAAGGAGGACGGAGGCTCAGACACTCGGAAGCCTTTGAGGCACTCCAGCAAA ENSTOOOOO25852 ACGGGCCGAAGGAGGACGGAGGCTTCAGACACTCGGAAGCCTTTGAGGCACTCCAGCAAA

U.S. Patent Jul. 7, 2015 Sheet 19 Of 28 US 9,075,045 B2

Figure 16 (B) (continued) DNA sequence alignment A: NM_006825 Reports Links Homo sapiens cytoskeleton-associated protein 4 (CKAP4), mRNA gi 1992O316 ref NM006825. 2 (1992.0316) B BCO829.72 Reports Order cDNA clone, Links Homo sapiens cytoskeleton-associated protein 4, mRNA (cDNA clone MGC: 995.54 IMAGE: 6084968), complete cds gi. 527892.52 gbBCO82972. 1 (52789252) C: BC094824 Reports Order cDNA clone, Links Homo sapiens cytoskeleton-associated protein 4, mRNA (cDNA clone MGC: 104815 IMAGE: 6374931) Complete cds gi. 631,02282 gbBC094824. 1 (631.02282) CLUSTAI, W (1.82) multiple sequence alignment

seqA ------GGGGGAGCCCCTGCAAGTTTCCCGGGCCGCGCGCCGCGC seqB ------AGCC - - - - - AA------TGGG ------GC seqC GCGTGCCGCTCGCCCAGTCCCGGGGGAGCCCCTGCAAGTTTCCCGGGCCGCGCGCCGCGC

start secA TCGCTCGCCTCCCAGCCCGCGGCCCGAGCCGCCGCCGCGCCCGCCATGCCCCGGCCAAA sedB ICGCTCGCCTCCCAGCCCGCGGCCCGAGCCGCCGCCGCGCCCGCCAGCCCTCGGCCAAA seqC TCGCTCGCCTCCCAGCCCGCGGCCCGAGCCGCCGCCGCGCCCGCCAGCCCTCGGCCAAA ...... kerk kirk k ......

segA CAAAGGGGCTCCAAGGGCGGCCACGGCGCCGCGAGCCCCTCGGAGAAGGGGCCCACCCG seq8 CAAAGGGGCTCCAAGGGCGGCCACGGCGCCGCGAGCCCCTCGGAGAAGGGTGCCCACCCG seqC CAAAGGGGCTCCAAGGGCGGCCACGGCGCCGCGAGCCCCTCGGAGAAGGGTGCCCACCCG ......

SeqA TCGGGCGGCGCGGATGACGGGCGAAGAAGCCGCCGCCGGCGCCGCAGCAGCCGCCGCCG seq8 TCGGGCGGCGCGGATGACGTGGCGAAGAAGCCGCCGCCGGCGCCGCAGCAGCCGCCGCCG seqC TCGGGCGGCGCGGAEGACGTGGCGAAGAAGCCGCCGCCGGCG ------......

seq.A. CCGCCCGCGCCGCACCCGCAGCAGCACCCGCAGCAGCACCCGCAGAACCAGGCGCACGGC seq8 CCGCCCGCGCCGCACCCGCAGCAGCACCCGCAGCAGCACCCGCAGAACCAGGCGCACGGC seqC ------

seqA AAGGGCGGCCACCGCGGCGGCGGCGGCGGCGGCGGCAAGTCCCCTCCTCCTCCTCCGCC seq8 AAGGGCGGCCACCGCGGCGGCGGCGGCGGCGGCGGCAAGCCTCCTCCTCCCCTCCGCC seqC ------

seqA CCGCCGCCGCTGCCGCCGCCGCCGCCCGTCCTCGGCGTCCTGCTCGCGCAGGCTCGGC seq8 TCCGCCGCCGCTGCCGCCGCCGCCGCCTCGTCCTCGGCGTCCIGCTCGCGCAGGCTCGGC seqC ------

sediA AGGGCGCTCAACTTTCCTTCTACCTCGCCCTGGTGGCGGCGGCCGCTTTCTCGGGCTGG seq8 AGGGCGCTCAACTTTCTCTTCTACCTCGCCCTGGTGGCGGCGGCCGCTTTCTCGGGCTGG seqC ------GCCGCTTTCTCGGGCTGG ......

secA TGCGTCCACCACGTCCTGGAGGAGGTCCAGCAGGTCCGGCGCAGCCACCAGGACTTCTCC seq8 TGCGTCCACCACGTCCTGGAGGAGGTCCAGCAGGTCCGGCGCAGCCACCAGGACTTCTCC seqC TGCGTCCACCACGTCCTGGAGGAGGCCAGCAGGTCCGGCGCAGCCACCAGGACTTCTCC k ......

seqA CGGCAGAGGGAGGAGCTGGGCCAGGGCTTGCAGGGCGTCGAGCAGAAGGTGCAGTCTTTG seqB CGGCAGAGGGAGGAGCGGGCCAGGGCTTGCAGGGCGTCGAGCAGAAGGTGCAGTCTTTG seqC CGGCAGAGGGAGGAGCTGGGCCAGGGCGCAGGGCGCGAGCAGAAGGGCAGCTG k ......

U.S. Patent Jul. 7, 2015 Sheet 22 of 28 US 9,075,045 B2

Figure 16 (E) (continued) seqA GTTGGGGGGACCCTTTTCTTTCTAGTTGTCTTTAAGGAAAATTAATTTTACTTTTTTTTT seqB ------AAAA------seqC ------AAAA------

seqA GTTCTGGCCGAAATTTTTATGAGATATCTCTCACTTGTCTTCCACTTTGAACCGGTTAA seq8 ------seqC ------

SeqA AGCTCATAGCTGTCAGCTCGAATGAGGAGGGGAGAAGCCCCTGGGTCTTTCTTTGAAAG seqB ------seqC ------

SeqA GAACCGCTGCTTGAGGGCTGCCTCCCTCATGGTGTGCGTGTCGTTCTCTCCTGACGCA seqB ------seqC ------

Seqa TCTGTGATATCAGAGGTAACTATGCAAAGCATCCAGGCGGTTCTGAATGTGAAGCACTAC seqes ------seqC ------

seqA ACCCAGCAGAGTCCCGGTGCCCTCTGTCCCCACTGCCGGCCCATGTCCTCTCTCCGGAGG seqB ------seqC ------

Seqa TCACCAAGGAATGCACAGGTTTCGACTACCAGAAAGGGGAGTCCTTGGGTTCTTTCAAAA seqB ------seqC ------

seqA AATTCGTGAGGAGAGCTGTCTACAGTGGAATAGGGGGTCTCCCTGGGGAATGCAGGCCAA seqB ------seqC ------

seqA GTCCITTTATTTTAACATGATGCCATGAAGAGGTTTGCCGCTGGGCAGCCCTGTCGGC seqb ------seqC ------

seqA AAGGAGCGTGCATACTGCGTTTGTGTAATTGTTTGCTGTATCTCCCTTCCCTCTGAGCTG seqB ------seqC ------

Seqa TATTGTTCTTTAATGGCTGTCTTGCCCTTCCAAAAAAAATTGAAAAAAAAAAAA seqb ------seqC ------U.S. Patent Jul. 7, 2015 Sheet 23 of 28 US 9,075,045 B2

Figure 17 17(A). Homo sapiens , reference assembly, complete Sequence gi89161190 refNC 000012.10|NC 000012 (Source) www.ncbi.nlm.nih.gov

105,157 K. i05,58 K. 105.59k 105,160 K. 105,6 K 105,162 K. 1956. 105,184 k 105,65K 105,166K

sixes -w---st C13D935. 80885. s

17B. (Source) genome.ewha.ac.kr/ECgene/ Gene Summary for H12c1364 - Gene Name : cytoskeleton-associated protel 4 - Gene Symbol: CKAP4 - Alias: AK223563 BC082972 CKAP4 NMOO6825 X6991 Op.63 AK123693 CLMP-63 P63 BC094824 Genomic Position: thr12; 105,155,782-105,222.87 bp. size: 66,205 . Clustering Summary for H2C13645 This gene Consisits of 327 sequences that include RefSeq, 7 mRNA, 319 EST sequences. According to ECgene analysis, this gene produces 10 transcript variants encoding 7 distinct proteins. . Clustering Summary for H2C3645 with High Confidence level A) This gene consisits of 277 sequences that include RefSeq, 7 mRNA, 269 EST sequences. According to ECgene analysis, this gene produces 9 transcript variants encoding 6 distinct proteins.

Transcripts go up

# a Representative Transcript, Re Refse. ::::::::::::::: ... -- . . . :::::::::::::::::::::::: Confidence level. A including RefSeq, if intil if single-exon;having mRNA and itsequence 3) . . ... Bintain initiekolai single-exonisequencer) 3'-'. :::::::::: ... Caltranscripts except Aid B.: ... 3"..:::: Franscript D Taip-r- conce d'sw rat ESTs "E. plei rRA, CDS eur 3'UTR H12C13S4S1R to dis1 A 1 3 fg 2 a 30gstp796 bp (265aa) 2.298 bp 1 bp H12C13S452 A 9 17 4 O 2.683 bp. 582 bp (194a3) 1930 bp 171 bp H12C13645.3i to ding A 1 218 45 3 265 hp 884 bp (228aa) 1930 hp bip "H13 in T. 1 92 19 3" in 2,725 hp E82 bp (134a) 1930 bp 21.4 bp TH2C135.55 coding A T 1 n 2 1578 hp. 428bp (142aa) 1,43 bp 1 bp. 2C134 Coding A 1 a En 2 2 1792 bp 428 bp (342a3) 1363 bp 1 bp. H2C138A57 A 1 o 8 C 1.195 bp 582 bp (194a3) 457 hp. 157 bp H2C1385.8 A. 1 n 7 - 3. 1,2R3hp 462 hp (154aa) $57 hp 35thp C1385.8 i A. 1 a i A. O 1,120 bp 582 bp (194aa) 138bp 400 bp U.S. Patent Jul. 7, 2015 Sheet 24 of 28 US 9,075,045 B2

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SAGE Carcer 18C, U.S. Patent Jul. 7, 2015 Sheet 25 of 28 US 9,075,045 B2

Figure 19

Orthologs for CKAP4 gene from 10 species (see top 5) Organismsit- Gene Locusf, Description isiNitaryHuman T.NCB accessions T. ------dog CKAP4' - cytoskeleton-87.04(n) C.------nu-na--- 481295 XM 538416.2 a------a------XP 538416.2 (Canis associated 8434(a) familiaris) i protein 4 in-e-r-la------arrechimpanzeeCKAP4' - cytoskeleton-98.05(n) in-waii-, 742720 XM 001161127.1 -,XP_001161127.1 -iti - (Pan associated 97.26(a) 1989) -- - Protein. ------cow CKAP4" - cytoskeleton- E. 515784XM 868540.2 XP873633.2 |--|--|-pent-----(Bos taurus): associated 84.69(a) rat Ckap4 - cytoskeleton-84.79(n) 362859 XM 3431893 XP-343190.1 i(Rattus : jassociated 83.97(a) Polis-a-la-Per-at-lars,use Clap4. 10' cytoskeleton-85.190) 216197' NM 175451.1'a NP780660.1--- (Musnusculus) Ckap4'. . ; . proteinasSociated 4 II.3.97(a) AC140333' AI152085 (seeI. all 24) . . . chicken CKAP4' -- cytoskeleton- 61.5(n) 418073 XM 416309.2 XP416309.2 (Gallus associated 55.47(a) gallus) protein 4 ETAzebrafish ----, zgc:85975------zge:85975------46.62(n) 406549 NMM- 213265.1------NP998.430.1 ------(Danio 25.89(a) rerio) d clawedAfrican frog Bassagiari." t" easo) passaga. (Xenopus I

laevis)------i------sa-YrWre-war------w------r-m------wv- - tropical BX707940.1 - - - 72.06(n) i BX70794.0.1 clawed frog; i (Silurana i :

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O 2). 300 40 SO 8. (s US 9,075,045 B2 1. 2 CELL-BASED DETECTION OF APF modifications, bladder training, and exercise, are other THROUGHTS INTERACTION WITH CKAP4 approaches to symptom relief. Surgical procedures such as FOR DAGNOSIS OF INTERSTITIAL fulguration and resection of ulcers, bladder augmentation, or CYSTITIS bladder removal (cystectomy) are a consideration only if all available treatments have failed and if the pain is disabling: BACKGROUND OF THE INVENTION however, most Surgeons are reluctant to operate because some patients still have symptoms after Surgery. 1. Field of Invention IC presents with symptoms that are identical to those This invention relates to an assay system designed to detect detected in patients with bacterial infections and other disor a protein biomarker in urine that is diagnostic for interstitial 10 cystitis (IC). The cytoskeletal associated protein 4 (CKAP4) ders of the urogenital tract (Table 1) (Wein et al., 1990). In is subcloned in frame with yellow-fluorescent protein (YFP) addition to the diseases listed in Table 1, differential diagnosis creating a fusion protein (CKAP4-YFP). CKAP4-YFP is includes several other possibilities, including overactive ectopically expressed in the human cervical carcinoma cell bladder (although these patients do not have bladder pain), line, HeLa, at the cell Surface, creating a cell-based system. 15 chronic urinary tract infection in anotherwise normal urinary Urine samples from patients who exhibit symptoms consis tract, Vulvodynia (or tender vulva) which may coexist with tent with IC are added to the cell-based system. The presence IC, and endometriosis which may also coexist with IC. These of a 9 amino acid glycopeptide, antiproliferative factor conditions are individually treated using different clinical (APF), in urine is unique to patients with IC. APF specifically approaches. Significantly, there is no commercially available binds to CKAP4-YFP at the cell surface. Binding of APF to diagnostic test for IC, making it difficult to distinguish it from CKAP4-YFP enables transit of CKAP4-YFP-APF to the other conditions with similar symptomology and difficult to nucleus. Transit of this protein complex to the nucleus is develop an appropriate treatment strategy. These gaps in measured using high-throughput immunofluorescence knowledge directly affect patient care: in the past, patients microscopy. Nuclear localization of the YFP signal is positive experienced an average lag time of five to seven years before for the presence of APF in urine and diagnostic for IC. The 25 they received a diagnosis of IC (Curhan et al., 1999). With cell-based diagnostic system is a significant and Surprising current prevalence estimates for IC in the managed care popu advance in diagnosis of IC and has commercial applications lation of 197/100,000 for women and 41/100,000 for men relevant to greater than 147/100,000 women and 41/100,000 (Clemens et al., 2005), hundreds of thousands of people would men who suffer from symptoms consistent with IC. have to be screened in order to differentiate IC from other 2. Description of Related Art 30 conditions (Clemens et al., 2005). At present the only avail Interstitial cystitis/painful bladder syndrome (IC/PBS) is a able diagnostic specific to IC is an ulcer of the bladder poorly understood, chronic, urinary bladder disease mani mucosa, which is called Hunner's ulcer. Unfortunately, this fested by bladder pain and increased frequency and urgency condition is not very helpful for the purposes of diagnosis of urination (Sant and Hanno, 2001). IC has no single, defin because it is found in 10% or fewer IC cases (Santand Hanno, able presentation, but is best viewed as a continuum extend 35 2001). Thus, there is a critical need for improved strategies to ing across decades of an individual’s life, beginning with accurately diagnose IC. mild, intermittent symptoms that become more severe and constant overtime. For many, pain is the most dominating and TABLE 1 debilitating symptom, becoming so severe as the disease Diseases that may mimic symptoms of IC progresses that it significantly affects patients’ quality of life 40 (Wein et al., 1990) (Marshall, 2003). As many as 1.3 million American men, Active genital herpes women, and children of all ages and races have IC; however, Vaginitis the disease is far more common in women (Clemens et al. Bladderstones 2007). Approximately 60/100,000 adult American women Bladder cancer 45 Cancer of the uterus, cervix, vagina, or urethra are affected, with more recent studies suggesting a Substan Urethral diverticulum tially higher prevalence (Curhan et al., 1999; Leppilahti et al. Radiation cystitis 2005). Indeed, the prevalence in the managed care population Cyclophosphamide cystitis was 197/100,000 for women and 41/100,000 for men when Bladder tuberculosis Neurogenic bladder diagnosed using ICD-9 code (Clemens et al., 2005). Because Drug effects: aspirin, NSAIDs, allopurinol the causes of IC are unknown, current treatments are aimed 50 only at providing symptom relief. Many patients have noted an improvement in Symptoms after bladder distention has Etiology of Interstitial Cystitis: Despite multiple hypoth been performed to diagnose the condition; however, research eses about the primary cause of IC, the underlying molecular ers are not sure why this helps. Bladder instillation (also mechanism of this disease remains completely undefined. called a bladder wash or bath) with dimethyl sulfoxide given 55 Active theories include increased permeability of the bladder every week or two for 6 to 8 weeks has also been noted to mucosa, abnormal neuronal function, mast cell activation, improve symptoms temporarily. Because DMSO passes into autoimmunity, infections, and toxic or antiproliferative Sub the bladder wall during this procedure, physicians believe that stances in the urine (Erickson et al., 1999); however, insuffi it may reach tissue more effectively to reduce inflammation, cient data exist to definitively establish their roles in the block pain, and also prevent muscle contractions that cause 60 pathology of IC. One of the most consistent findings in blad pain, frequency, and urgency. Pentosan polysulfate sodium der mucosal biopsies from IC patients has proven to be thin (the first oral drug developed for IC: Elmiron) given orally ning or erosion of the bladder epithelium (Keay, 2008: Leiby (100 mg three times a day) has improved symptoms in 30% of etal, 2007). It is speculated that normal repair of the damaged patients. Bacillus Calmette-Guerin (BCG), a common intra bladder epithelium does not occur inpatients who develop IC: vesicular agent used in bladder cancer, has also been shown to 65 thus, urine contents, such as potassium, can leak into the improve symptoms (Peters et al., 1998). Electrical nerve bladder interstitium, possibly leading to mast cell activation stimulation and lifestyle interventions, including dietary and histamine release. In response, C-fiber nerves become US 9,075,045 B2 3 4 activated, triggering the release of Substance P as well as (Keay et al., 2004; Keay et al., 2000). The peptide sequence of immunogenic and allergic responses. These events are APF is identical to residues 541-549 of the 6th transmem thought to lead to progressive bladder injury, which may brane domain of Frizzled 8, a Wnt ligand receptor. The gly promote spinal cord changes in Some patients, causing cosyl moiety of APF consists of sialic acid C-2.3 linked to chronic neuropathic pain (Hanno, 2007). Research studies galactose B1-3-N-acetylgalactosamine, which is C.-O-linked have demonstrated abnormal cell signaling in IC bladder to the N-terminal threonine residue of the nonapeptide. APF epithelial cells as compared to controls. The expression of has been shown to profoundly inhibit the proliferation of several proteins, including group D-related human leukocyte normal bladder epithelial, bladder carcinoma, and cervical antigen (HLA-DR) and inter-cellular adhesion molecule 1 adenocarcinoma cells in vitro (Keay et al., 2004; Keay et al. ICAM-1, the secreted proteins IL-1, TNFC., and various epi 10 2000). Furthermore, APF can induce multiple changes in the thelial growth factors (i.e., FIB-EGF, EGF, and IGF1) have pattern of cellular including decreased pro been shown to have altered expression in bladder biopsies duction of HB-EGF and increased production of E-cadherin, and/or explanted primary bladder epithelial cells from IC resulting in a more differentiated bladder epithelial cell phe patients (Keay, 2008). In addition, increased paracellular per notype (Keay et al., 2000; Keay et al., 2003). APF was also meability and abnormalities in tight junction protein expres 15 recently determined to decrease tightjunction protein (Zonula sion (i.e., Zonula occludens-1 ZO-1, occludin, and claudin occludens-1 and occludin) production and increase paracel 1, 4, and 8) have been demonstrated in explanted bladder lular permeability of normal bladder epithelial cell monolay epithelial cells grown for IC patient biopsies as compared to ers similar to changes seen in cells from patients with IC in cells from normal controls (Zhang et al., 2005; Keay et al. vitro (Zhang et al., 2005). This accumulation in urine of a 2003a; Zhang et al., 2007). These abnormalities may be bioactive factor, capable of altering the behavior of urothelial related to altered bladder epithelial cell differentiation in IC cells, is consistent with the clinical observation of epithelial patients, with increased E-cadherin expression and decreased thinning and denudation observed in IC bladder tissue. expression of ZO-1 and uroplakin III, and altered cytokeratin The potency of APF (EC50 in the picomolar range), its gene expression in vivo and/or in vitro (Keay et al., 2003a; varied effects on bladder epithelial cell protein expression Zhang et al., 2007; Hauser et al., 2008; Southgate et al., 2007; 25 and proliferation, and the requirement for a hexosamine Slobodov et al., 2004: Laguna et al., 2006; Keay, 2008). Blad galactose disaccharide linked in a specific alpha configura der epithelial cells from IC patients also exhibit profoundly tion to the backbone peptide for activity, all indicate that decreased proliferation (Keay et al., 2003b), decreased APF's effects are mediated by binding to and activating a expression of cyclin D1 and INK (Keay et al., 2003a), and receptor. increased paracellular permeability in vitro in the absence of 30 In 2006, Conrads et al identified cytoskeletal associated exogenous serum or growth factors as compared to cells from protein 4 (CKAP4) (also known as p63, CLIMP-63, ERGIC normal controls (Zhanget al., 2005). Collectively, these find 63), as a high affinity receptor for APF (Conradset al., 2006). ings provide additional indirect evidence for altered epithelial CKAP4 has also been identified as a functional cell surface cell signaling in IC. receptor for tissue plasminogen activator (tPA) in Smooth Current Approaches to IC Diagnosis: The diagnosis of IC 35 muscle cells (Razzaq et al., 2006) and for surfactant protein A in the general population is based on the presence of pain (SP-A) in rat type II pneumocytes (Gupta et al., 2006). related to the bladder, usually accompanied by frequency and CKAP4 is a nonglycosylated, reversibly palmitoylated, type urgency to urinate, in the absence of other diseases that could II transmembrane protein. At its NH terminus, CKAP4 has a cause the symptoms. Diagnostic tests that help rule out other 106-amino acid long cytosolic tail, a single transmembrane disease include urinalysis, urine culture, cystoscopy, biopsy 40 domain, and a large extracytoplasmic domain of 474 amino of the bladder wall, distention of the bladderunder anesthesia, acids. Original studies of CKAP4 described it as an endoplas urine cytology, and laboratory examination of prostate secre mic reticulum (ER) resident protein (Schweizer et al., 1994). tions (Sant and Hanno, 2001). Multiple urine markers for IC Subsequently, it was shown to aid in the anchoring of rough have been described including antiproliferative factor (APF), ER to microtubules in epithelial cells (ie, COS and HeLa) EGF, IGF binding protein-3, and interleukin (IL)-6, which are 45 (Klopfenstein et al., 1998). This function requires a direct significantly increased in IC and HB-EGF, cyclic guanosine interaction between the cytoplasmic N-terminal tail of the monophosphate, and methylhistamine, which are signifi protein to microtubules and is regulated by phosphorylation cantly decreased in IC. In a study that compared these mark (Vedrenne et al., 2005). ers, APF was found to have the least overlap in the IC and A system for detecting APF bioactivity utilizes H-thymi control groups (Erickson et al., 2002). APF was originally 50 dine incorporation to measure the cellular proliferation of purified from the urine of IC patients, and bioactivities attrib cultured normal bladder epithelial cells (explanted from uted to APF include suppression of urothelial cell prolifera human bladder tissue) following exposure of the cells to tion; increases in transcellular permeability; lowering of the patient urine. This bioassay does not discriminate from other expression of proteins that form intercellular junctional com factors present in urine that have also been shown to regulate plexes; and reduction in the production of HB-EGF from 55 cellular proliferation (e.g., heparin binding epidermal growth urothelial cells (Keay et al., 2004). APF activity is detectable factor-like growth factor (HB-EGF), epidermal growth factor in the urine of approximately 95-97% of IC patients who (EGF), interleukin-1 (IL-1) and insulin-like growth factor 1 fulfill the symptomatic, exclusionary, and cystoscopic (IGF1), etc.) A system for detecting APF by generating spe NIDDK criteria for IC (Keay et al., 2001; Keayetal, 1996) and cific antibodies against this 9 amino acid glycopeptides is 94% of patients who fulfill symptomatic and exclusionary 60 problematic. It's size of 9 amino acids indicate it is not a good criteria alone (Keay et al., 2007). The specificity of APF for immunogen as this is the minimal size epitope for any anti urine from IC patients (vs. normal controls or patients with a body to recognize and well below minimal sized polypeptides variety of other urogenital disorders (Keay et al., 2001)) sug used to generate specific antibodies. Further, developing a gests that it is useful as a diagnostic marker for IC. specific antibody against APF is also problematic given that Potential Role of APF Interstitial Cystitis: APF is a low 65 its 9 amino acids are identical to sequences foundina receptor molecular weight sialoglycopeptide secreted specifically protein called Frizzled 8, presenting the potential to cross from bladder epithelial cells in patients suffering from IC react with a larger protein that shares this sequence. A second US 9,075,045 B2 5 6 system for detecting APF is the focus of the present invention, of heterozygosity on chromosomal band 8p22 has been includes measuring its presence based on bioactivity, a cell shown to be a common event in some epithelial tumors, based system that expresses a specific receptor for APF and pointing toward the likelihood that the region harbors poten upon presentation of urine samples to the cell based system, tial tumor suppressor genes (Emi et al., 1993: Fujiwara et al., binding of APF induces translocation of the receptor to the 1993: Ichii et al., 1993; Ohata et al., 1993) Because DHHC2 nucleus. The receptor protein is fused to a fluorescent indica has no other known signaling properties beyond palmitoyla tor signaling protein. The presence of the fluorescent signal in tion, knowledge of its target Substrates in a cancer cell line the nucleus is quantified by high-throughput microscopy. could yield significant clues about its role in metastasis and Translocation of the fluorescent signal into the nucleus is tumor Suppression. In previous work, we used a novel, pro diagnostic for the presence of APF and for IC. 10 teomic method called PICA to identify the target substrates of In addition, Palmitoylation is the posttranslational addition DHHC2 in HeLa cells, a cervical adenocarcinoma cell line. of the 16-carbon palmitate group to specific cysteine residues We determined that cytoskeletal associated protein 4 of proteins (Smotrys and Linder, 2004) via a labile thioester (CKAP4, also known as p63, ERGIC-63, and CLIMP-63) is bond. Unlike otherforms of lipidation, such as myristoylation a principle, physiologically important substrate of DHHC2 and prenylation, palmitoylation is reversible which allows for 15 (Zhang et al., 2008). dynamic regulation of protein-membrane interactions, traf CKAP4 is a reversibly palmitoylated, type II transmem ficking between membrane compartments (Wedegaertner brane protein that has been shown to anchor rough ER to and Bourne, 1994; Jones et al., 1997: Moran et al., 2001; microtubules in epithelial cells (ie, COS and HeLa) (Sch Zacharias et al., 2002), and synaptic plasticity (el-Husseini weizer et al., 1993a; Schweizer et al., 1993b; Schweizer et al., Ael and Bredt, 2002). For many years it was believed that 1994; Schweizer et al., 1995a. Vedrenne and Hauri, 2006). palmitoylation occurred primarily by autocatalytic mecha This function requires a direct interaction between the cyto nisms (BiZZoZero et al., 1987; Bano et al., 1998); however, the plasmic N-terminal tail of the protein to microtubules and is recent discovery of a family of palmitoyl acyl transferase regulated by phosphorylation of three critical serine residues (PAT) enzymes that catalyze protein palmitoylation has (Klopfenstein et al., 1998). More recently, CKAP4 has been reversed this notion, expanding the complexity of the mecha 25 identified as a functional cell surface receptor for antiprolif nisms by which palmitoylation is regulated (Lobo et al., 2002: erative factor (APF) (Conrads et al., 2006), a low molecular Roth et al., 2002: Fukata et al., 2004; Linder and Deschenes, weight, Frizzled-8 protein-related sialoglycopeptide secreted 2007). from bladder epithelial cells in patients suffering from the PATs are encoded by the ZDHHC gene family and are chronic, painful bladder disorder, interstitial cystitis (IC) characterized by an Asp-His-His-Cys motif (DHHC) within a 30 (Keay et al., 2000; Keay et al., 2004a). APF profoundly inhib cysteine-rich domain (CRD). The DHHC and CRD domains its normal bladder epithelial cell growth (Keay et al., 1996; are essential for palmitoyl acyl transferase activity (Roth et Keay et al., 2000; Keay et al., 2004a). APF also inhibits the al., 2002: Fukata et al., 2004; Sharma et al., 2008). Twenty proliferation of bladder carcinoma cells and HeLa cells in three genes encoding proteins with DHHC-CRD domains vitro with an IC50 of ~1 nM (Keay et al., 2004a: Conradset have been identified in mouse and human databases (Fukata 35 al., 2006; Keay et al., 2006). Binding of APF to CKAP4 et al., 2004). Of these, at least seven have been shown to be results in inhibition of cellular proliferation and altered tran associated with human disease: DHHC8 with schizophrenia scription of at least 13 genes known to be involved in the (Mukai et al., 2004); DHHC17/HIP14 with Huntington's dis regulation of proliferation and tumorigenesis (including ease (Yanai et al., 2006); DHHC15 and DHHC9 with E-cadherin, vimentin, cyclin D1, p53 and ZO-1) (Keay et al., X-linked mental retardation (Mansouri et al., 2005; Raymond 40 2003: Conrads et al., 2006: Kim et al., 2007). et al., 2007); and DHHC2, DHHC9, DHHC17, and DHHC11 The present invention is directed to the effects of reduced with cancer (Oyama et al., 2000; Duckeret al., 2004; Mansilla CKAP4 palmitoylation on APF-mediated signaling by silenc et al., 2007; Yamamoto et al., 2007). In many of these ing the expression of DHHC2 with targeted siRNA. Our data examples, the absence of PAT expression and Subsequent show that DHHC2-mediated palmitoylation of CKAP4 is a failure to palmitoylate target Substrates is the underlying 45 critical event regulating CKAP4 subcellular distribution, problem. APF-stimulated changes in cellular proliferation and gene Although now recognized as a PAT, DHHC2 was previ expression, as well as APF-independent changes in cellular ously known as ream for reduced expression associated with migration. metastasis. As the name Suggests, this gene was first identified All references cited herein are incorporated herein by ref because its expression level was consistently and signifi 50 erence in their entireties. cantly reduced in clonal murine colorectal adenocarcinoma cell lines with high metastatic potential, but not in clonal lines BRIEF SUMMARY OF THE INVENTION derived from the same tumor that did not metastasize (Tsuruo et al., 1983; Oyama et al., 2000). It was concluded that ream The invention provides a nucleic acid reporter gene con expression is inversely related to the metastatic potential of a 55 struct comprising: (a) a promoter; (b) a nucleic acid sequence cell, leading to speculation that this gene normally suppresses selected from the group consisting of a CKAP4 gene, frag one or more of the processes by which cancer cells escape ments thereof, and variants thereof, and (c) a reporter gene. from blood vessels, invade into and proliferate in a target The invention provides a nucleic acid comprising a reporter organ, and induce angiogenesis and form metastatic foci. gene operatively linked to the gene encoding CKAP4, frag Human ZDHHC2 maps to a region of (p21.3- 60 ments thereof, and variants thereof. The invention further 22) that is frequently deleted in many types of cancer, includ provides the nucleic acid, wherein the encoded CKAP4, frag ing colorectal (Fujiwara et al., 1993: Ichii et al., 1993: Fuji ment thereof, or variant thereof is capable of binding APF. wara et al., 1994) hepatocellular carcinoma (Erniet al., 1993; The invention further provides the nucleic acid, wherein the Fujiwara et al., 1994), non-small cell lung (Fujiwara et al., encoded CKAP4, fragment thereof, or variant thereof is 1993; Ohata et al., 1993), and cancers of the breast (Yaremko 65 capable of binding APF and is capable of translocation to the et al., 1996; Anbazhagan et al., 1998), urinary bladder nucleus. The invention further provides the nucleic acid, fur (Knowles et al., 1993), and prostate (Bova et al., 1993). Loss ther comprising a protein synthesis termination signal and/or US 9,075,045 B2 7 8 a polyadenylation signal downstream of the reporter gene gene, fragments thereof, and variants thereof, wherein the sequence. The invention further provides the nucleic acid, encoded CKAP4 gene, fragment thereof, or variant thereof is wherein the reporter gene encodes a fluorescent polypeptide capable of binding APF and is capable of translocation to the selected from the group consisting of a green fluorescent nucleus. The invention further provides the stably transfected protein (GFP), an enhanced GFP (EGFP), a destabilized host cell, wherein the cell is selected from the group consist enhanced GFP (d2EGFP), a blue fluorescent protein (BFP), a ing of VERO, HeLa, CHO, COS, W 138, BHK, HepG2,3T3, cyan fluorescent protein (CFP) a yellow fluorescent protein RIN, MDCK, A549, PC12, K562 and 293 cells. The invention (YFP), or a red fluorescent protein (RFP including DsRed), a provides a host cell comprising a CKAP4 inducible promoter fluorescent protein of any spectral characteristics from any region and a reporter gene operatively linked to the CKAP4 organism, a fluorescent protein that is a monomer, and a 10 inducible promoter region. The invention further provides the fluorescent protein that is a fused dimer, and combinations host cell, wherein the cell is selected from the group consist thereof. The invention further provides the nucleic acid, ing of VERO, HeLa, CHO, COS, W 138, BHK, HepG2,3T3, wherein the gene product of the reporter gene is a protein RIN, MDCK, A549, PC12, K562 and 293 cells. The invention whose enzymatic activity is detectable. The invention further provides a stably transfected host cell line comprising a provides the nucleic acid, wherein the gene product is LUC 15 CKAP4 inducible promoter region and a reporter gene opera (luciferase), AP (alkaline phosphatase), SEAP (secretory tively linked to the CKAP4 inducible promoter region. The alkaline phosphatase) or CAT (chloramphenicol acetyltrans invention further provides the stably transfected host cell, ferase). The invention further provides the nucleic acid, wherein the cell is selected from the group consisting of wherein the reporter protein is an immunologically detectable VERO, HeLa, CHO, COS, W138, BHK, HepG2, 3T3, RIN, protein. The invention further provides the nucleic acid, MDCK, A549, PC12, K562 and 293 cells. wherein the reporter protein is a selection marker. The inven The invention provides an isolated protein encoded by a tion further provides the nucleic acid, wherein the vectors nucleic acid construct comprising: (a) a promoter; (b) a further comprises a sequence encoding an affinity tag. The nucleic acid sequence selected from the group consisting of a invention further provides the nucleic acid, wherein the affin CKAP4 gene, fragments thereof, and variants thereof, and (c) ity tag is selected from the group consisting of a polyhistidine 25 a reporter gene. The invention further provides the protein, tag, polyarginine tag, glutathione-S-transferase, maltose further comprising a protein synthesis termination signal and/ binding protein, staphylococcal protein A tag, an EE-epitope or a polyadenylation signal downstream of the reporter gene tag, histidine tag, tag-100, c-myc, E2. V5 epitope tag, a 6-His sequence. The invention provides an isolated protein com epitope tag, a c-myc tag, a Flag tag, a GFP tag, a GST tag, a prising a reporter gene operatively linked to the gene encod HA tag, a luciferase tag, a Protein C tag, an S-tag, a T7 tag, a 30 ing CKAP4, fragments thereof, and variants thereof. thioredoxin tag, a VSV-g tag. His-tag, a Flag-tag, an HA-tag. The invention further provides the protein, wherein the a myc-tag, and other epitope tag. reporter gene encodes a fluorescent polypeptide selected The invention provides a nucleic acid comprising: (a) a from the group consisting of a green fluorescent protein CKAP4 inducible promoter region; (b) a reporter gene opera (GFP), an enhanced GFP (EGFP), a destabilized enhanced tively linked to the CKAP4 inducible promoter region. The 35 GFP (d2EGFP), a blue fluorescent protein (BFP), a cyan invention further provides the nucleic acid, wherein the fluorescent protein (CFP) a yellow fluorescent protein (YFP), reporter gene encodes a fluorescent polypeptide selected or a red fluorescent protein (RFP including Dsked), a fluo from the group consisting of a green fluorescent protein rescent protein of any spectral characteristics from any organ (GFP), an enhanced GFP (EGFP), a destabilized enhanced ism, a fluorescent protein that is a monomer, and a fluorescent GFP (d2EGFP), a blue fluorescent protein (BFP), a cyan 40 protein that is a fused dimer, and combinations thereof. fluorescent protein (CFP) a yellow fluorescent protein (YFP), The invention provides an isolated protein comprising a or a red fluorescent protein (RFP including Dsked), a fluo reporter gene operatively linked to the gene encoding rescent protein of any spectral characteristics from any organ CKAP4, fragments thereof, and variants thereof. The inven ism, a fluorescent protein that is a monomer, and a fluorescent tion further provides the protein, wherein the reporter gene is protein that is a fused dimer, and combinations thereof. The 45 selected from the group consisting of a green fluorescent invention further provides the nucleic acid, wherein the gene protein (GFP), an enhanced GFP (EGFP), a destabilized product of the reporter gene is a protein whose enzymatic enhanced GFP (d2EGFP), a blue fluorescent protein (BFP), a activity is detectable. The invention further provides the cyan fluorescent protein (CFP) a yellow fluorescent protein nucleic acid, wherein the gene product is LUC (luciferase), (YFP), or a red fluorescent protein (RFP including DsRed), a AP (alkaline phosphatase), SEAP (secretory alkaline phos 50 fluorescent protein of any spectral characteristics from any phatase) or CAT (chloramphenicol acetyltransferase). The organism, a fluorescent protein that is a monomer, and a invention further provides the nucleic acid, wherein the fluorescent protein that is a fused dimer, and combinations reporter protein is an immunologically detectable protein. thereof. The invention further provides the protein, wherein The invention further provides the nucleic acid, wherein the the gene product of the reporter gene is a protein whose reporter protein is a selection marker. 55 enzymatic activity is detectable. The invention further pro The invention provides a host cell comprising a reporter vides the protein, wherein the gene product is LUC (lu gene operatively linked to a nucleic acid sequence selected ciferase), AP (alkaline phosphatase), SEAP (secretory alka from the group consisting of a CKAP4 gene, fragments line phosphatase) O CAT (chloramphenicol thereof, and variants thereof, wherein the encoded CKAP4, acetyltransferase). The invention further provides the protein, fragment thereof, or variant thereof is capable of binding APF 60 wherein the reporter protein is an immunologically detectable and is capable of translocation to the nucleus. The invention protein. The invention further provides the protein, wherein further provides the host cell, wherein the cell is selected from the reporter protein is a selection marker. The invention fur the group consisting of VERO, HeLa, CHO, COS, W138, ther provides the protein, wherein the encoded CKAP4, frag BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562 and 293 ment thereof, or variant thereof is capable of binding APF. cells. The invention provides a host cell line stably transfected 65 The invention further provides the protein, wherein the with a reporter gene operatively linked to a nucleic acid encoded CKAP4, fragment thereof, or variant thereof is sequence selected from the group consisting of a CKAP4 capable of binding APF and is capable of translocation to the US 9,075,045 B2 10 nucleus. The invention further provides the protein, wherein population of host cells comprising a nucleic acid sequence each of the expression vectors further comprises a sequence selected from the group consisting of a CKAP4 gene, frag encoding an affinity tag. The invention further provides the ments thereof, and variants thereof, wherein the encoded protein, wherein the affinity tag is selected from the group CKAP4, fragment thereof, or variant thereof is capable of consisting of a polyhistidine tag, polyarginine tag, glu binding APF and is capable of translocation to the nucleus and tathione-S-transferase, maltose binding protein, Staphylococ a reporter gene; (b) providing a biological sample from a cal protein A tag, an EE-epitope tag, histidine tag, tag-100, patient; (c) contacting the first host cell population with the c-myc, E2, V5 epitope tag, a 6-His epitope tag, a c-myc tag, a biological sample; (d) providing a control sample; (e) con Flagtag, a GFP tag, a GST tag, a HA tag, a luciferase tag, a tacting the second host cell population with the control Protein C tag, an S-tag, a T7 tag, a thioredoxin tag, a VSV-g 10 tag. His-tag, a Flag-tag, an HA-tag, a myc-tag, and other sample: (f) isolating nuclei from the first and second host cell epitope tag. populations to provide a first and second nuclei population; The invention provides a kit comprising the nucleic acid, (g) measuring reporter gene expression in the first nuclei and instructions for use. The invention provides a kit com population: (h) measuring reporter gene expression in the prising the host cell, and instructions for use. 15 second nuclei population; and (i) contacting the first nuclei The invention provides a method for diagnosis of IC dis population with an antibody that binds the protein encoded by ease in a patient by detecting the presence of APF in a bio the reporter gene to form a first antibody complex: (i) con logical sample comprising: (a) providing a first and a second tacting the second nuclei population with an antibody that population of host cells comprising a nucleic acid sequence binds the protein encoded by the reporter gene to form a selected from the group consisting of a CKAP4 gene, frag second antibody complex; and (k) providing a detection anti ments thereof, and variants thereof, wherein the encoded body; (1) contacting the detection antibody with the first anti CKAP4, fragment thereof, or variant thereof is capable of body complex; (m) contacting the detection antibody with the binding APF and is capable of translocation to the nucleus and second antibody complex; (n) detecting the presence of the a reporter gene; (b) providing a biological sample from a detection antibody that bound to the first and second antibody patient; (c) contacting the first host cell population with the 25 complex; and (o) assessing the reporter gene expression in the biological sample; (d) providing a control sample; (e) con first antibody complex relative to the reporter gene expression tacting the second host cell population with the control in the second antibody complex, wherein detecting an sample: (f) isolating nuclei from the first and second host cell increase in said reporter gene expression in the first antibody populations to provide a first and second nuclei population; complex identifies the presence of APF in the biological (g) measuring reporter gene expression in the first nuclei 30 population: (h) measuring reporter gene expression in the sample, thereby indicating IC disease. The invention further second nuclei population; and (i) assessing the reporter gene provides a method wherein the biological sample is a member expression in the first nuclei population relative to the selected from the group consisting of whole blood, serum, reporter gene expression in the second nuclei population, plasma, cerebrospinal fluid, saliva, urine, spinal fluid, Syn wherein detecting an increase in said reporter gene expres 35 ovial fluid, amniotic fluid and cranial fluid, and lymphocyte or sion in the first nuclei population identifies the presence of cell culture Supernatants. APF in the biological sample, thereby indicating IC disease. The invention provides a method for diagnosis of IC dis The invention further provides a method wherein the nucleic ease in a patient by detecting APF protein in a biological acid sequence is integrated into a chromosome of the host sample comprising: (a) providing a first and a second popu cells or maintained episomally. 40 lation of host cells comprising a nucleic acid sequence The invention further provides a method wherein the host selected from the group consisting of a CKAP4 gene, frag cells are a mammalian cell population. The invention further ments thereof, and variants thereof, wherein the encoded provides a method wherein the mammalian cell population is CKAP4, fragment thereof, or variant thereof is capable of a human cell population. The invention further provides a binding APF and is capable of translocation to the nucleus and method wherein the reporter polypeptide is a fluorescent 45 a reporter gene; (b) providing a biological sample from a polypeptide. The invention further provides a method patient; (c) contacting the first host cell population with the wherein the fluorescent polypeptide is selected from the biological sample; (d) providing a control sample; (e) con group consisting of a green fluorescent protein (GFP), an tacting the second host cell population with the control enhanced GFP (EGFP), a destabilized enhanced GFP sample: (f) isolating nuclei from the first and second host cell (d2EGFP), a blue fluorescent protein (BFP), a cyan fluores 50 populations to provide a first and second nuclei population; cent protein (CFP) a yellow fluorescent protein (YFP), or a (g) measuring reporter gene expression in the first nuclei red fluorescent protein (RFP including DsRed), a fluorescent population: (h) measuring reporter gene expression in the protein of any spectral characteristics from any organism, a second nuclei population; and (i) contacting the first nuclei fluorescent protein that is a monomer, and a fluorescent pro population with an antibody that binds the protein encoded by tein that is a fused dimer, and combinations thereof. The 55 the reporter gene to form a first antibody complex: (i) con invention further provides a method wherein the gene product tacting the second nuclei population with an antibody that of the reporter gene is a protein whose enzymatic activity is binds the protein encoded by the reporter gene to form a detectable. The invention further provides a method wherein second antibody complex; and (k) providing a detection anti the gene product is LUC (luciferase), AP (alkaline phos body to the first antibody complex; (1) providing a detection phatase), SEAP (secretory alkaline phosphatase) or CAT 60 antibody to the second antibody complex; (m) performing gel (chloramphenicol acetyltransferase). The invention further electrophoresis on the first antibody complex; (n) performing provides a method wherein the reporter protein is an immu gel electrophoresis on the second antibody complex; (o) nologically detectable protein. The invention further provides detecting the presence of the detection antibody that bound to a method wherein the reporter protein is a selection marker. the first and second antibody complex; and (p) assessing the The invention provides a method for diagnosis of IC dis 65 reporter gene expression in the first nuclei population relative ease in a patient by detecting the presence of APF in a bio to the reporter gene expression in the second nuclei popula logical sample comprising: (a) providing a first and a second tion, wherein detecting an increase in said reporter gene US 9,075,045 B2 11 12 expression in the first nuclei population identifies the pres potential changes. The invention further provides a method ence of APF in the biological sample, thereby indicating IC wherein the method of measurement is generated/detected disease. following APF binding due to a change in the conformation of The invention provides an isolated antibody or fragment the receptor, a change in protein-protein interactions, a thereof which specifically binds a polypeptide selected from 5 change in membrane-protein interactions or other. The other the group consisting of CKAP4 gene, fragments thereof, and interacting partners being labeled with an appropriate fluoro variants thereof. The invention further provides an antibody phore to serve as a resonance energy donor or acceptor that or fragment thereof wherein said polypeptide is glycosylated. appropriately complements the donor or acceptor fluorophore The invention further provides an antibody or fragment that is attached to CKAP4. thereof which is polyclonal. The invention further provides an 10 The invention further provides a method wherein the sig antibody or fragment thereof which is selected from the group nals from FRET, BRET, LRET and LET may be detected consisting of a) achimericantibody; b) a Fab fragment; and c) either intramolecularly, where the donor and acceptor fluo a F(ab) fragment. The invention further provides an antibody rophore are part of the same sensor molecule (e.g., CKAP4) or fragment thereof which is labeled. The invention further or intermolecularly where the donor and acceptor are physi provides an antibody or fragment thereof wherein the label is 15 cally associated with two separate molecules, on of which selected from the group consisting of: a) an enzyme; b) a may be CKAP4, the other any molecule that interacts with fluorescent label; and c) a radioisotope; d) and epitope tag. CKAP4 in an APF-dependent manner. The invention further provides an antibody or fragment The invention provides a method of detecting an agent that thereof which specifically binds to said polypeptide in a West binds and/or modulates the activity of CKAP4, the method ern blot. The invention further provides an antibody or frag comprising: (a) contacting a CKAP4 protein, fragments ment thereof wherein the epitope tag is selected from the thereof, and variants thereof, wherein the CKAP4 protein is group consisting of a polyhistidine tag, polyarginine tag. capable of binding APF, with an APF protein, fragments glutathione-S-transferase, maltose binding protein, staphylo thereof, and variants thereof in the presence of a candidate coccal protein A tag, an EE-epitope tag, histidine tag, tag agent under conditions, which in the absence of the test agent, 100, c-myc, E2. V5 epitope tag, a 6-His epitope tag, a c-myc 25 permit the binding of the APF polypeptide to the CKAP4 tag, a Flag tag, a GFP tag, a GST tag, a HA tag, a luciferase polypeptide; and (b) determining whether the candidate agent tag, a Protein C tag, an S-tag, a T7 tag, a thioredoxin tag, a is capable of modulating the interaction between the CKAP4 VSV-g tag. His-tag, a Flag-tag, an HA-tag, a myc-tag, and polypeptide and the APF polypeptide. other epitope tag. The invention further provides an antibody The invention further provides a method wherein the APF or fragment thereof which specifically binds to said polypep 30 polypeptide is a polypeptide comprising: (a) the sequence of tide in an ELISA. The invention further provides an antibody APF; or (b) a sequence which is at least 70% identical to APF or fragment thereof which specifically binds to said polypep and which binds to and activates a signaling activity of tide in a competitive-binding assay. The invention further CKAP4; or is a fragment of APF which binds to and activates provides an antibody or fragment thereof which specifically a signaling activity of CKAP4. The invention further provides binds to said polypeptide in a radioimmunoassay. The inven 35 a method wherein the CKAP4 polypeptide is a polypeptide tion provides an isolated cell that produces the antibody or comprising: (a) the sequence of CKAP4; (b) a sequence fragment thereof. The invention provides a hybridoma that which is at least 70% identical to CKAP4 over its entire length produces the antibody of fragment thereof. and functionally equivalent to CKAP4; or (c) a fragment of The invention provides a method for diagnosis of IC dis CKAP4 which is functionally equivalent to CKAP4. The ease in a patient by detecting the presence of APF in a bio 40 invention further provides a method wherein the candidate logical sample comprising: (a) providing a detectably labeled agent is a polypeptide, an antibody or antigen-binding frag first CKAP4 protein, fragments thereof, and variants thereof, ment thereof, a lipid, a carbohydrate, a nucleic acid, a small wherein the CKAP4 protein is capable of binding APF; (b) molecule, or a chemical compound. The invention further providing a detectably labeled second CKAP4 protein, frag provides a method wherein step (b) comprises monitoring ments thereof, and variants thereof, wherein the CKAP4 pro 45 binding of the CKAP4 polypeptide to the APF polypeptide. tein is capable of binding APF; (c) providing a biological The invention further provides a method wherein the binding sample from a patient; (d) contacting the detectably labeled of the CKAP4 polypeptide to the APF polypeptide is moni first CKAP4 protein with the biological sample to provide a tored using label displacement, Surface plasmon resonance, first test sample; (e) providing a control sample: (f) contacting fluorescence resonance energy transfer, fluorescence quench the detectably labeled second CKAP4 protein with the con 50 ing or fluorescence polarization. The invention further pro trol sample to provide a second test sample; (g) measuring the vides a method wherein the APF polypeptide is detectably label in the first test sample: (h) measuring the label in the labelled. The invention further provides a method wherein the second test sample; and (i) assessing the label in the first test APF polypeptide is detectably labelled with a moiety is a sample relative to the label in the second test sample, wherein radioisotope, a fluorophore, a quencher of fluorescence, an detecting an increase in the first test sample identifies the 55 enzyme, an affinity tag or an epitope tag. The invention fur presence of APF in the biological sample, thereby indicating ther provides a method wherein step (b) comprises monitor IC disease. The invention further provides a method wherein ing the signaling activity of the CKAP4 polypeptide. The the method of measurement is selected from the group con invention further provides a method wherein the signaling sisting of fluorescence resonance electron transfer (FRET), activity is monitored by measurement of guanosine nucle lanthanide resonance electron transfer (LRET), fluorescence 60 otide binding, GTPase activity, adenylate cyclase activity, cross-correlation spectroscopy, fluorescence quenching, cyclic adenosine monophosphate (cAMP), Protein Kinase C fluorescence polarization, flow cytometry, Scintillation proX activity, phosphatidylinositol breakdown, diacylglycerol, imity, luminescence resonance energy transfer, direct inositol, triphosphate, intracellular calcium, MAP kinase quenching, ground-state complex formation, chemilumines activity or reporter gene expression. The invention further cence energy transfer, bioluminescence resonance energy 65 provides a method wherein step (b) comprises monitoring the transfer, excimer formation, colorimetric Substrates detec translocation activity of the CKAP4 polypeptide. The inven tion, phosphorescence, electro-chemical changes, and redox tion further provides a method wherein the CKAP4 polypep US 9,075,045 B2 13 14 tide is expressed on a cell. The invention further provides a peptide as compared to a level of binding of the APF to the method wherein the cell is a mammalian cell. CKAP4 expressed in cytoplasmic membrane in the absence The invention provides a method for assessing activity of a of the test peptide indicates the test peptide inhibits the bind candidate compound in interfering with binding of an APF ing of an APF to CKAP4 expressed in a cytoplasmic and/or protein with a CKAP4 protein expressed in a eukaryotic of a eukaryotic cell. The invention further cytoplasmic and/or cell membrane, comprising: assessing, in provides a method wherein the peptide is a variant of APF. the presence and absence of a candidate compound, binding The invention further provides a method wherein the eukary of a polypeptide comprising an APF protein with a CKAP4 otic cell is a mammalian cell. The invention further provides protein expressed in a eukaryotic cytoplasmic and/or cell a method wherein the polypeptide comprises a detectable membrane; wherein reduced binding in the presence of the 10 label. The invention further provides a method wherein the candidate compound relative to a level of binding in the detectable label is a fluorescent label or a radioisotope. The absence of the candidate compound indicates activity of the invention further provides a method wherein the fluorescent candidate compound in interfering with binding of APF pro label is a green fluorescent protein. tein to the CKAP4 protein expressed in a eukaryotic cytoplas The invention provides a method for diagnosis of IC dis mic and/or cell membrane. The invention further provides a 15 ease in a patient by detecting the presence of APF in a bio method wherein the assessing is performed extracellularly. logical sample comprising: (a) providing a detectably labeled The invention further provides a method wherein the assess first CKAP4 protein, fragments thereof, and variants thereof, ing is performed in intact cells. The invention further provides wherein the CKAP4 protein is capable of binding APF; (b) a method wherein the cell membranes are a cytoplasmic providing a detectably labeled second CKAP4 protein, frag and/or cell membrane preparation, and wherein said assess ments thereof, and variants thereof, wherein the CKAP4 pro ing comprises determining the binding of the APF to the cell tein is capable of binding APF; (c) providing an isosteric membrane preparation in the presence of a candidate com agent which binds CKAP4; (d) contacting the isosteric agent pound and in the absence of a candidate compound, wherein to the first CKAP4 protein to provide a first CKAP4/agent a decrease in the binding of the APF to the cell membrane sample; (e) contacting the isosteric agent to the second preparation in the presence of the compound as compared to 25 CKAP4 protein to provide a second CKAP4/agent sample: (f) binding in the absence of the compound identifies the com providing a biological sample from a patient, (g) contacting pound as interfering with binding of the APF to the cytoplas the detectably labeled first CKAP4/agent sample with the mic and/or cell membranes of the eukaryotic cell. The inven biological sample to provide a first test sample: (h) providing tion further provides a method wherein the method is a control sample: (i) contacting the detectably labeled second performed intracellularly in a eukaryotic cytoplasmic and/or 30 CKAP4/agent sample with the biological sample to provide a cell. The invention further provides a method wherein the second test sample; () measuring the label in the first test APF is coupled to a detectable label and the intracellular sample; (k) measuring the label in the second test sample; and distribution of the label is assessed in the presence of a can (1) assessing the label in the first test sample relative to the didate compound, wherein the candidate compound is iden label in the second test sample, wherein detecting an increase tified as interfering with binding of the HCV nonstructural 35 in the first test sample identifies the presence of APF in the protein to a eukaryotic cytoplasmic and/or cell membrane biological sample, thereby indicating IC disease. when its presence results in an intracellular distribution of the The invention provides a method to detect CKAP4/APF label which does not show binding to cytoplasmic and/or cell endocytosis/endocytic vesicles comprising: (a) providing a membranes in comparison to the distribution of the label in first and a second population of host cells comprising a the absence of the compound. The invention further provides 40 nucleic acid sequence selected from the group consisting of a a method wherein the detectable label is green fluorescent CKAP4 gene, fragments thereof, and variants thereof, protein. The invention further provides a method wherein the wherein the encoded CKAP4, fragment thereof, or variant eukaryotic cell is a mammalian cell. The invention further thereof is capable of binding APF and is capable of translo provides a method wherein the non-structural protein is cation to the nucleus and a reporter gene; (b) providing a NS4B. The invention further provides a method wherein the 45 biological sample from a patient; (c) contacting the first host non-structural protein is NS5A. The invention further pro cell population with the biological sample; (d) providing a vides a method wherein the candidate compound is a peptide. control sample; (e) contacting the second host cell population The invention further provides a method wherein the candi with the control sample: (f) isolating vesicles from the host date compound is a modified version of APF that binds to cells to provide a first and second vesicle population; (g) CKAP4 but does not activate the receptor. The invention 50 detecting the presence of the reporter gene in the first and further provides a method wherein the candidate compound is second vesicle populations; and (h) assessing the reporter a modified version of endogenous APF. The invention further gene expression in the first vesicle population relative to the provides a method wherein the candidate compound is a second vesicle population, wherein detecting an increase in difference in the sugar moiety that modifies the N-terminus of said reporter gene expression in the first vesicle population APF. The invention further provides a method wherein the 55 identifies the presence of APF in the biological sample, candidate compound is or the addition of a modified C-ter thereby indicating IC disease. The invention further provides minus of APF. a method wherein the nucleic acid sequence is integrated into The invention provides a method of identifying a peptide a chromosome of said population of cells or maintained epi that inhibits the binding of an APF to CKAP4 peptide somally. The invention further provides a method wherein the expressed in a cytoplasmic and/or cell membrane of a eukary 60 population of cells is a mammalian cell population. The otic cell, comprising contacting a polypeptide comprising an invention further provides a method wherein the mammalian APF protein with CKAP4 peptide expressed in cytoplasmic cell population is a human cell population. The invention and/or cell membranes of a eukaryotic cell in the presence and further provides a method wherein the reporter polypeptide is absence of a test peptide; and assessing binding of the APF of a fluorescent polypeptide. The invention further provides a the polypeptide to the cytoplasmic and/or cell membranes; 65 method wherein the fluorescent polypeptide is selected from wherein a decreased level of binding of the APF to the cyto the group consisting of a green fluorescent protein (GFP), an plasmic and/or cell membrane in the presence of the test enhanced GFP (EGFP), a destabilized enhanced GFP US 9,075,045 B2 15 16 (d2EGFP), a blue fluorescent protein (BFP), a cyan fluores first test sample identifies the presence of APF in the biologi cent protein (CFP) a yellow fluorescent protein (YFP), or a cal sample. The invention further provides a method wherein red fluorescent protein (RFP including DsRed), a fluorescent the reporter polypeptide is a fluorescent polypeptide. The protein of any spectral characteristics from any organism, a invention further provides a method wherein the fluorescent fluorescent protein that is a monomer, and a fluorescent pro polypeptide is selected from the group consisting of a green tein that is a fused dimer, and combinations thereof. The fluorescent protein (GFP), an enhanced GFP (EGFP), a desta invention further provides a method wherein the gene product bilized enhanced GFP (d2EGFP), a blue fluorescent protein of the reporter gene is a protein whose enzymatic activity is (BFP), a cyan fluorescent protein (CFP) a yellow fluorescent detectable. The invention further provides a method wherein protein (YFP), or a red fluorescent protein (RFP including the gene product is LUC (luciferase), AP (alkaline phos 10 DsRed), a fluorescent protein of any spectral characteristics phatase), SEAP (secretory alkaline phosphatase) or CAT from any organism, a fluorescent protein that is a monomer, (chloramphenicol acetyltransferase). The invention further and a fluorescent protein that is a fused dimer, and combina provides a method wherein the reporter protein is an immu tions thereof. The invention further provides a method nologically detectable protein. wherein the gene product of the reporter gene is a protein The invention provides a method of detecting a APF pro 15 whose enzymatic activity is detectable. The invention further tein in a biological sample comprising: (a) providing a first provides a method wherein the gene product is LUC (lu and a second population of host cells comprising a nucleic ciferase), AP (alkaline phosphatase), SEAP (secretory alka acid sequence selected from the group consisting of a CKAP4 line phosphatase) or CAT (chloramphenicol acetyltrans gene, fragments thereof, and variants thereof, wherein the ferase). The invention further provides a method wherein the encoded CKAP4 is capable of binding APF and is capable of reporter protein is an immunologically detectable protein. translocation to the nucleus and a reporter gene; (b) providing The invention provides a kit for detecting interstitial cys a biological sample from a patient; (c) contacting the first host titis in a patient, by detecting the presence of APF in a bio cell population with the biological sample; (d) providing a logical sample comprising said kit comprising: (a) at least one control sample; (e) contacting the second host cell population detection reagent which is specific for APF present in a fluid with the control sample: (f) isolating nuclei from the first and 25 sample collected from the patient Suspected of having the second host cell populations to provide a first and second interstitial cystitis, wherein said at least one detection reagent nuclei population; (g) measuring reporter gene expression in is adapted to detect a threshold level of said APF, said thresh the first nuclei population: (h) measuring reporter gene old level adapted to indicate a presence of interstitial cystitis; expression in the second nuclei population; and (i) contacting and (b) instructions for using said at least one detection the first nuclei population with an antibody that binds the 30 reagent to evaluate interstitial cystitis in the patient. The protein encoded by the reporter gene to form a first antibody invention further provides a kit wherein the fluid sample is complex; () contacting the second nuclei population with an urine. The invention further provides a kit whereina detection antibody that binds the protein encoded by the reporter gene reagent is selected from the group consisting of antigens; to form a second antibody complex; and (k) providing a haptens; monoclonal and polyclonal antibodies; natural and detection antibody to the first antibody complex: (1) providing 35 synthetic mono-, oligo- and polysaccharides; lectins; avidin a detection antibody to the second antibody complex; (n) and streptavidin; biotin; growth factors; hormones; receptor detecting the presence of the detection antibody that bound to molecules; and combinations thereof. The invention further the first and second antibody complex; and (o) assessing the provides a kit wherein the detection reagent is selected from reporter gene expression in the first nuclei population relative monoclonal and polyclonal antibodies. The invention further to the reporter gene expression in the second nuclei popula 40 provides a kit wherein the detection reagent comprises an tion, wherein detecting an increase in said reporter gene antibody or antibody fragment. The invention further pro expression in the first nuclei population identifies the pres vides a kit wherein the detection reagent comprises CKAP4 ence of APF in the biological sample, thereby indicating IC protein, fragments thereof, and variants thereof, wherein the disease. CKAP4 protein is capable of binding APF. The invention The invention provides a method to measure APF in a 45 further provides a kit wherein said kit comprises a detector biological sample comprising: (a) providing a first reporter device having a Support in communication with at least one of vector comprising a promoter region, wherein the promoter said at least three detection reagents. The invention further region is responsive to CKAP4/APF binding, a nucleic acid provides a kit wherein said kit comprises a dipstick, a Swab, sequence selected from the group consisting of a CKAP4 and a container. The invention further provides a kit wherein gene, fragments thereof, and variants thereof, and a reporter 50 the detection reagents are attached to a detectable label. The gene and CKAP4 protein, fragments thereof, or variants invention further provides a kit further comprising one or both thereof to provide a first assay Sample; (b) providing a second of a positive and a negative control. reporter vector comprising a promoter region, wherein the The invention provides a method of screening for intersti promoter region is responsive to CKAP4/APF binding, a tial cystitis in a patient, said method comprising the steps of nucleic acid sequence selected from the group consisting of a 55 (a) obtaining a fluid sample from a patient; (b) applying the CKAP4 gene, fragments thereof, and variants thereof, and a sample to a detector device, wherein the detector device com reporter gene and CKAP4 protein, fragments thereof, or vari prises at least one detection reagent which is specific for APF, ants thereof to provide a second assay sample; (c) providing a wherein said at least one detection reagent is adapted to detect biological sample; (d) contacting the first assay sample with a threshold level of APF, said threshold level correlated with the biological sample to provide a first test sample; (e) pro 60 a presence of interstitial cystitis; (c) ascertaining the thresh viding a control sample: (f) contacting the second assay old level of APF present in said sample, wherein if the con sample with the control sample to provide a second test centration each of said APF exceeds the threshold level, then sample; (g) measuring reporter gene activity in the first test this is a positive screen for interstitial cystitis. The invention sample: (h) measuring reporter gene activity in the second test further provides a method wherein the fluid sample is urine. sample; and (i) comparing reporter gene activity in the first 65 The invention further provides a method wherein said ascer test sample compared to the second test sample, wherein taining is performed by an enzyme-linked immunosorbent detecting an increase in said reporter gene expression in the assay. The invention further provides a method wherein the US 9,075,045 B2 17 18 detection reagent is selected from the group of species con exponential decay. The half life was calculated to be 13.87 sisting of antigens; haptens; monoclonal and polyclonal anti hours (R2=0.998, n=3 error bars=SE). (6B) The relative bodies; natural and synthetic mono-, oligo- and polysaccha abundance of DHHC2 mRNA in siRNA-transfected cells rides; lectins: avidin and streptavidin; biotin; growth factors; versus mock transfected control cells was determined after 96 hormones; receptor molecules; and combinations thereof. hours by qRT-PCR. DHHC2 expression was reduced -84% The invention further provides a method wherein the detec with siRNA knockdown. (6C) CKAP4 interacts with CFP tion reagent is selected from monoclonal and polyclonal anti DHHC2. CFP-tagged DHHC2 was immunoprecipitated from bodies. The invention further provides a method wherein said transiently-transfected HeLa cells using an anti-GFP mAb reagents comprise an antibody or antibody fragment. The (see Materials and Methods). Western blot analysis with an invention further provides a method wherein the detection 10 anti-CKAP4 antibody detected a robust band at ~63 kDa reagent comprises CKAP4 protein, fragments thereof, and characteristic of CKAP4 in lysates from cells transfected with variants thereof, wherein the CKAP4 protein is capable of CFP-DHHC2 but not in lysates from mock transfected cells. binding APF. The invention further provides a method Exposure time was 15 seconds. wherein said detector device is a dipstick comprising at least FIG. 7 shows that the DHHC2-mediated palmitoylation of one detection reagent which is specific for APF. 15 CKAP4 on cysteine 100 regulates its trafficking from the ER to the PM. (7A) Mock-transfected or ZDHHC2 siRNA-trans BRIEF DESCRIPTION OF SEVERAL VIEWS OF fected HeLa cells were grown on LabTek multiwell glass THE DRAWINGS slides (Nalge Nunc), fixed, and incubated with a mAb G1/296 against CKAP4 (“anti-CLIMP-63, diluted 1:100, Alexis FIG. 1 is a diagram showing the cloning strategy of creat Biochemicals) followed by a TRITC-labeled, goat anti ing CKAP4-YFP. To monitor visually, by fluorescence mouse secondary antibody (diluted 1:1000, Jackson Immu microscopy, the subcellular distribution of CKAP4, wild noResearch Laboratories) or with a pAb against calreticulin type CKAP4 was fused to the C-terminus of monomeric (diluted 1:1000, Abcam) followed by a FITC-labeled, goat yellow fluorescent protein (CKAP4-YFP). The plasmid anti-rabbit secondary antibody (diluted 1:1000, Invitrogen). pcDNA3.1 is used for subcloning. Here, CKAP4 is subcloned 25 Controls included cells processed without primary and/or in frame with YFP sequences which exist at the 3' end of the secondary antibodies. Images were acquired using a Nikon multiple cloning site. This results in a coding sequence that TE2000 epifluorescence microscope with NIS Elements can direct expression of CKAP4-YFP fusion protein. Tran Software and overlayed using Adobe Photoshop software. In Scription of this gene in cells is driven by the strong promoter HeLa cells with reduced DHHC2 expression, CKAP4 local sequence, CMV. 30 ization is restricted to the ER (white arrows). (7B) HeLa cell FIG. 2 shows HeLa cells expressing CKAP4-YFP in the lines stably expressing CKAP4WTV5 or the palmitoylation absence and presence of synthetic APF (Peptides Interna incompetent mutant, CKAP4 C100S-V5, were grown on tional, Louisville Ky.). In HeLa cells transiently transfected LabTek multiwell glass slides (Nalge Nunc), fixed, and with the CKAP4-YFP construct, CKAP4-YFP localization immunolabeled with a FITC-conjugated mAb antibody was observed on the plasma and perinuclear membranes simi 35 against the V5 epitope (1:500: Invitrogen). CKAP4WT was lar to endogenous CKAP4 expression (Zhang et al., 2008: expressed on the plasma membrane and perinuclear mem Planey et al., 2008). The application of synthetic APF to these branes, whereas CKAP4C100S expression was restricted to transiently transfected cells promotes rapid nuclear localiza the ER. Epifluorescence images in 7B were made with a tion of CKAP4:YFP. 100x, 1.45 NA oil immersion objective (Nikon). FIG.3 shows endogenous CKAP4 localization in the pres 40 FIG. 8 shows that the palmitoylation of CKAP4 by ence and absence of APF, which is consistent with the appear DHHC2 is required for APF-stimulated translocation of ance of CKAP4-YFP expressed in HeLa cells (Conrads et al. CKAP4 to the nucleus. Mock-transfected or DHHC2 siRNA 2006; Razzaq et al., 2006: Zhang et al., 2008: Planey et al. transfected HeLa cells were treated with APF (20 nM) for 48 2008: Gupta et al., 2006: Bates et al., 2008). hours. Cells were fixed and incubated with a mAb G1/296 FIG. 4 confirms the results of FIG. 2 and FIG. 3 using a 45 against CKAP4 (“anti-CLIMP-63, Alexis Biochemicals) biochemical approach where the nuclear fraction of a cell is followed by a TRITC-labeled, goat anti-mouse secondary separated from the cytoskeletal fraction. The proteins associ antibody (Jackson ImmunoResearch Laboratories). CKAP4 ated with each fraction are resolved by SDS polyacrylamide translocated to the nucleus and nucleolus in HeLa cells gel electrophoresis (SDS-PAGE), proteins transferred to treated with APF (red arrows); however, this translocation nitrocellulose by western transfer and the nitrocellulose 50 was blocked in APF-treated HeLa cells transfected with probed with anti-CKAP4 antibodies using western blot DHHC2 siRNA. Epifluorescence images were made with a analysis. CKAP4 abundance is increased in nuclear fractions 60x1.45 NA oil immersion objective (Nikon). from APF treated HeLa cells. Application of synthetic APF to FIG.9 shows the siRNA-mediated knockdown of DHHC2 these cells resulted in a 5-fold increase in the abundance of blocks the antiproliferative response of HeLa and NB cells to CKAP4 in the nucleus versus untreated cells. 55 APF. (9A) HeLa cells were electroporated with DHHC2 FIG. 5 is a depiction of cells expressing CKAP4-YFP double-stranded siRNA (solid circles) or with nonsense (panel A). Urine samples from patients displaying symptoms siRNA (open circles) as a control for nonsequence-specific consistent with IC are added to the cells (panel B). If APF is effects on Day 1, serum-starved on Day 2, and varying con present in the patient urine sample, CKAP4-YFP moves to centrations of APF or control peptide (0.25-250 nM) were the nucleus (panel C) while, patient samples that do not have 60 added to the medium on Day 3; cells were then cultured for an APF will revealCKAP4-YFP at the cell membrane (panel D). additional 48 hours under conditions of serum starvation. FIG. 6 shows the half-life of DHHC2 mRNA. (6A) The (9B) NB epithelial cells were electroporated with DHHC2 abundance of DHHC2 mRNA after siRNA-mediated double-stranded siRNA (solid circles) or with nonsense DHHC2 knockdown was measured by qRT-PCR at 0, 12, 24, siRNA (Solid triangles) as a control for nonsequence-specific 48, 72 and 96 hours following siRNA oligo transfection. 65 effects on Day 1, serum-starved on Day 2, and varying con These experiments were run in triplicate. The data were centrations of APF or control peptide (0.25-250 nM) were approximated most accurately using a non-linear, first-order added to the medium on Day 3; cells were then cultured for an US 9,075,045 B2 19 20 additional 48 hours under conditions of serum starvation. For equal loading of protein, membranes were stripped and rep both cell types, cellular proliferation was assessed by inhibi robed with a mAb to B-actin (1:5000; Sigma). Proteins were tion of 3H-thymidine incorporation. Each data point repre visualized by enhanced chemiluminescence and Subsequent sents the mean and standard deviation of three independent exposure to film (BioMaxAR, Kodak). (12B) The signal experiments. Data are presented as percent inhibition of 5 intensity for vimentin was quantified by densitometry using 3H-thymidine incorporation compared to controls. The abil Image.J., normalized to the corresponding band for 3-actin, ity of APF to block proliferation was inhibited in the presence and reported as the fractional abundance of the control of DHHC2 siRNA for all APF concentrations except 250 nM (mock-transfected cells). Results shown are representative of for HeLa cells, or 25 and 250 nM for NB cells. three independent experiments that gave similar results. FIG. 10 shows APF-mediated changes in gene expression 10 FIG. 13 shows the Stable CKAP4 C100S expression are dependent on palmitoylation of CKAP4 by DHHC2 in NB increases the migratory rate of HeLa cells. HeLa cells stably cells. Primary NB epithelial cells were electroporated with transfected with CKAP4C100S, WT CKAP4, or parental DHHC2 double-stranded siRNA or with nonsense siRNA on controls were seeded in fibronectin coated, 6-well plates and Day 1, serum-starved on Day 2, and 2.5 nM APF or control grown to confluence. To permit measurement of migration in peptide were added to the medium on Day 3; cells were then 15 the absence of proliferation, cells were treated with AraC (5 cultured for an additional 48 hours under conditions of serum ug/ml. Sigma), blocking further cell division. After 8 hours in starvation. Expression of ZO-1, vimentin, and E-cadherin AraC, a line of adherent cells was scraped from the bottom of mRNA was assessed by quantitative real-time PCR as each well with a p-200 pipette tip to generate a “wound.” described in the Experimental Procedures. (10A, 10B) APF (13A) Cells were allowed to migrate into the wound for 24 alone or in the presence of nonsense siRNA reduced ZO-1 hours, and the extent of migration into the region from which and vimentin mRNA levels by ~.93% and -97%, respectively. cells had been scraped was determined from consecutive DHHC2 knockdown blocked this APF-stimulated reduction images of the same field of view taken at 3-hour intervals. in ZO-1 and vimentin mRNA levels. (10C) APF alone or in Cells were photographed using a Nikon Eclipse TE2000-U the presence of nonsense siRNA dramatically increased microscope under 20x magnification and phase contrast. E-cadherin mRNA levels, an effect that was also blocked by 25 (13B) The cell-free area introduced by each wound was mea DHHC2 knockdown. ZO-1, vimentin, and E-cadherin Sured using Image.J and converted to a percentage of area at mRNA levels were measured in triplicate runs and quantified time 0 plotted against time. These data were fit to a monoex by normalization to mRNA levels for D-actin using real-time ponential decay curve (Microcal Origin, Northampton PCR analysis software from Applied Biosystems. The error in Mass.). The T'/2 rate for migration of cells into the wound was the normalized, relative abundance of each mRNA species 30 calculated to be 2.3 hours for CKAP4C100S cells (red, open was propagated forward from the standard deviation of the circles) and 5.7 hours for WT CKAP4 cells (green squares); mean Ct value for each of the experimental samples and the parental HeLa cells did not migrate into the wound during the actin control. course of these experiments (horizontal blue diamonds across FIG. 11 shows the APF-mediated changes in protein the top of the graph). expression are dependent on palmitoylation of CKAP4 by 35 FIG. 14 shows that the palmitoylation of CKAP4 by DHHC2 in NB cells. Primary NB epithelial cells were trans DHHC2 is required for APF-mediated signaling. (14A) APF fected with DHHC2 double-stranded siRNA on Day 1, inhibits cellular proliferation and alters the expression of a serum-starved on Day 2, and 2.5 nM APF or control peptide specific set of genes by binding to its receptor, CKAP4 (Keay were added to the medium on Day 3; cells were then cultured et al., 2003; Zhanget al., 2005; Kim et al., 2007). APF binds for an additional 48 hours under conditions of serum starva 40 to CKAP4 with high affinity and this binding has been shown tion. FIG. 11A—ZO-1 and vimentin protein expression was to be necessary for mediation of APF-related signaling events analyzed by SDS-PAGE followed by Western blotting with (Conrads et al., 2006). CKAP4 is also one substrate (of an antibodies to ZO-1 (220 kDa) and vimentin (57 kDa) as unknown number) of the palmitoyl acyl transferase, DHHC2 described in Experimental Procedures. To assess equal load (Zhang et al., 2008). (14B) siRNA-mediated knockdown of ing of protein, membranes were stripped and reprobed with a 45 ZDHHC2 (the gene encoding the PAT, DHHC2) mRNA mAb to B-actin (1:5000; Sigma). Proteins were visualized by expression (see FIG. 6 half life or bar graph), prevents enhanced chemiluminescence and Subsequent exposure to CKAP4 trafficking from the ER to the PM (see FIGS. 7 and 8) film (BioMax AR, Kodak). FIG. 11B. The signal intensities and in doing so, inhibits APF binding to CKAP4. Conse for ZO-1 was quantified by densitometry using Image.J. nor quently, the inhibitory effect of APF on cellular proliferation malized to the corresponding band for B-actin, and reported 50 (see FIG.9) and APF-mediated changes in gene transcription as the fractional abundance of the control (mock-transfected (see FIGS. 10-12) are blocked. While it is likely that other cells). FIG. 11C. The signal intensities for vimentin were substrates of DHHC2 exist, APF mediated changes in cellular quantified by densitometry using Image.J., normalized to the proliferation and gene expression appear to be regulated by, corresponding band for 3-actin, and reported as the fractional DHHC2-mediated palmitoylation of CKAP4. abundance of the control (mock-transfected cells). Results 55 FIG. 15 shows multiple alignment of CKAP4 proteins that shown are representative of three independent experiments result from alternative splicing. FIGS. 15A, 15B, and 15C that gave similar results. show (SEQID NO: 11), (SEQID NO: 12), (SEQID NO: 13), FIG. 12 shows the APF-mediated changes in vimentin (SEQID NO: 17), (SEQID NO: 18), (SEQ ID NO: 19). protein expression are dependent on palmitoylation of FIG.16 shows multiple sequence alignment of CKAP4 and CKAP4 by DHHC2 in HeLa cells. HeLa cells were trans 60 variants. FIGS. 16A, 16B, 16C, 16D, and 16E show (SEQID fected with DHHC2 double-stranded siRNA, nonsense NO: 14), (SEQID NO:15), (SEQID NO: 16), (SEQID NO: siRNA, or mock-transfected and cultured for 48 hours. Cells 20), (SEQID NO: 21), (SEQID NO: 22). were then serum-starved, and the indicated cultures incubated FIG. 17 shows the detailed intron/exon structure of the with APF (20 nM) for an additional 48 hours. (12A) Expres gene encoding CKAP4. FIG. 17A shows the structure of sion of vimentin protein was analyzed by SDS-PAGE and 65 Homo sapiens chromosome 12 at 12q23.3 and the splice Western blotting with a mAb antibody against Vimentin (57 variants and expressed sequence tags (ESTs) known to exist. kDa) as described in Experimental Procedures. To assess FIG. 17B shows the gene summary for CKAP4. The gene is US 9,075,045 B2 21 22 comprised of multiple exons. Alternative splicing of these calculated to cause each molecule to emit 1,000 photons/s at exons may produce unique proteins that retain their ability to time-0 before photobleaching. The inset is an expansion of bind to and transduce the known effects of APF. the first 60s of bleaching. FIG. 18 shows the expression analyses of CKAP4 in a broad array of tissues and disease states. FIG. 18A 5 DETAILED DESCRIPTION OF PREFERRED GeneNote Expression array images. FIG. 18B GNF Nor EMBODIMENTS OF THE INVENTION mal Expression array images. FIG. 18C GNF Cancer Expression array images. Experimental tissue vectors: Dupli The invention provides, for example, a cell-based system cate measurements were obtained for twelve normal human whereby CKAP4, the receptor for APF, may be expressed at tissues (out of 28 tissues shown) hybridized against Affyme 10 the cell membrane as a yellow fluorescent protein (YFP) trix GeneChips HG-U95A-E (GeneNote data) and for 22 fusion protein (CKAP4-YFP). Urine samples of patients with normal human tissues hybridized against HG-U133A (GNF symptoms consistent with IC may be applied to the cell-based Symatlas data **). The intensity values (shown on the y-axis) system. The presence of APF in the urine sample is detected were first averaged between duplicates, then probeset values when it binds to the CKAP4-YFP receptor protein. Binding of 15 APF to CKAP4-YFP directs the receptor protein to translo were averaged per gene, global median-normalized and cate from the cell periphery (cellular plasma membrane) to scaled to have the same median of about 70 (half-way the nucleus. between GeneNOte and GNF medians). Available at GNF In another aspect, this invention is designed to detect APF Symatlas, HG-U133A expression data for 18 NCI60 cancer in urine samples. This result, combined with patient symp cell lines was processed and added to the display (a single toms would be diagnostic for IC and obviate the need for measurement taken; normalized according to the GNF patients to undergo current diagnostic procedures whereby Symatlas normal data). the patient is put under local anesthesia and an invasive pro FIG. 19 shows orthologs of the human CKAP4 gene. The cedure is performed to measure loss of thickening of the table presents the following columns: bladder wall. Thus, this invention would obviate the risks of Organism—The names of the homologous species, using anesthesia and invasive procedures and would also cost con both Scientific and popular terminology. 25 siderably less. Gene—The symbol for the gene in the homologous spe In another aspect, this invention is designed to distinguish C1GS. IC from other conditions whose symptoms overlap with IC. Locus—The position of the gene in the homologous spe Distinguishing one condition from another allows for a more cies. directed therapeutic approach for patients with these symp Description—Its description. 30 toms. If the urine sample where negative for APF, diagnostic Human Similarity. The percent similarity to the human procedures that measure bacteria in urine (e.g., urinalysis, gene, followed either by (n) where the comparison was urine culture; cytology); that test for bladder carcinoma (cys based on nucleic acids or (a) for amino acid based com toScopy, bladder biopsy, imaging tests ICT scan, MRI, Sono parisons. gram, intravenous pyelogram, bone scan, chest X-ray); over active bladder (urinalysis, postvoid residual Volume, simple NCBI accessions—links to the sequences for the gene in 35 cystometry, urodynamic testing, uroflowmetry, cystometro NCBI databases including GenBank and Entrez, Gene. gram, cystoscopy, and imaging tests X-ray, ultrasound), and Superscripts represent the source from which this data endometriosis (laparoscopy, histology) could then focus was extracted. If a '-' follows the superscript for patient treatment using therapeutic strategies that target these HomoloGene, it means that data for this species exists other conditions. only in the older version of HomoloGene, which used 40 This invention represents a significant advance for the unfinished genomes and where the homologs found diagnosis of IC. The invention is non-invasive and does not might not be true orthologs. The source of this informa require anesthesia, indicating it is safer. The invention is tion: www..org/info.shtml#ort estimated to cost a consumer S500 compared to current inva FIG. 20 shows T. Sapphire mCrange FRET. Emission spec sive diagnostic procedures which cost $10,000.00. This is a tra for 400 nm excitation for a zinc-finger fused with mCr 45 significant savings for consumers and insurance companies. ange on its N terminus and T-Sapphire on its C terminus. This invention represents a significant advance in that it is Emission in the presence of 1 mM EDTA in zinc-free buffer much faster to diagnosis than current procedures (30 minutes is represented by the green line, and emission in the presence compared to 1 week). This invention represents a Surprising of 1 mM ZnCl2 is represented by the orange line. advance in that the biological activity of a biomarker for IC FIG.21 shows discrimination of E. coli transfected with six 50 can be measured accurately and rapidly in a visual, high throughput system, improving diagnosis and treatment of IC. different fluorescent proteins. Bacteria separately trans From an epidemiological standpoint, the ease (requires only formed with different fluorescent proteins were mixed and the Submission of a urine sample) and availability of the assay analyzed by flow cytometry for a total of about 10,000 events. will mean that 1) many more patients that are Suspected of Excitation was at 514 nm from an argon-ion laser. Emissions having IC are more likely to be tested. It is possible that, due E1, E2, and E3 were simultaneously collected with 540-560 55 to the current difficulty in diagnosis, patients with milder or nm, 564-606 nm, and 595-635 nm bandpass filters respec shorter term symptoms are not likely to be tested. The tively. Normalized fluorescence F, were calculated as E/ increase in the number of patients Suspected of having IC that (mean of all E. values) for i=1, 2, 3. The root mean-square would be tested with this CKAP4:YFP assay may in time intensity (ordinate) was defined as (F/+F22+F/JII2, while the show that many more people have IC than previously angle sum (abscissa) was tan-I(FilE1)+tan-I(F31F2) in radi 60 believed. 2) The use of this test will more effectively exclude ans. Each dot represents a cell. Control runs with pure popu patients with IC symptoms that do not have the disease. The lations verified that the dots represent GFP-expressing cells, improvement in the delineating patient populations with pre dots represent Citrine YFP mBanana, mOrange, mStraw viously indiscernible symptom boundaries will also improve berry, and mOherry. our understanding of the epidemiology of IC and diseases that FIG. 22 shows photobleaching curves for new RFP vari 65 have similar symptomology. ants. Curves for mRFP1 and EGFP are included for compari From the symptomological standpoint, this assay will pro son. All curves are normalized to illumination intensities vide a concentration of APF in the urine of patients which can US 9,075,045 B2 23 24 then be correlated to other factors such as age of onset, dura any gene that expresses a detectable gene product Such as tion of time since the onset of symptoms, severity of symp RNA or protein. Therefore, expression of the gene product toms and so forth. With a sufficient number of patients diag can be determined using Northern or Western blot assays. nosed in a database, a much clearer understanding of the Alternatively, the gene product can be selected because it is progression of the disease can be obtained. easily measured. Examples of reporter genes encoding more The assay itself is conceptually simple. The metric for easily measured gene products include, but are not limited to, diagnosis is the absence or presence of CKAP4:YFP fluores alkaline phosphatase, luciferase, B-galactosidase, chloram cence in the nucleus following the application of patienturine phenicol acetyltransferase (CAT), lux operon, green fluores to the CKAP4:YFP reporter cells in culture; the nucleus being cent protein (GFP), and/or fluorescent protein variants. The defined by labeling with one of many fluorescent dyes that 10 alkaline phosphatase gene product is secreted and therefore bind nuclear DNA with exquisite specificity. Such dyes have may be very easily measured in the conditioned culture no spectral overlap with the fluorescence associated with medium. CKAP4:YFP. There are many ways to determine the absence or presence of CKAP4:YFP in the nucleus ranging from The assay may be run simultaneously with a second control qualitative, simple visual inspection by a trained professional assay wherein the control recombinant cell does not contain a to a quantitative, morphometric instrument—vision analysis 15 APF-responsive protein, such as, for example CKAP4. The on a microscope without any unintended analyst biases. An assay may be run simultaneously with a second control assay alternative approach to using a stably-expressed, fluorescent wherein the control sample does not contain APF, but the reporter to measure APF in the urine is to use immunolabeling recombinant cell contains an APF responsive protein, such as, of endogenous CKAP4. The metric in this case would remain for example CKAP4. The assay may be run simultaneously the same-abundance of CKAP4 in the nucleus. The labor with a control set of APF standards to generate a standard involved in immunodetection is somewhat greater than it curve from which sample concentrations of APF can be quan would be using CKAP4:YFP but may offer an advantage of titated. long-term stability of the cellular response to APF. Nucleic Acids Analysis by an unbiased instrument with a robust assay has A “APF responsive protein’ is a protein that is capable of the most potential to provide the highest density of data. 25 binding APF, wherein the activity of the APF-responsive pro However, alternative methods exist to make the same mea tein is altered in response to the binding, for example CKAP4. surement. One of these is the measurement of CKAP4 abun In one embodiment, the protein can be a cell Surface protein dance in the nucleus by Western blot following biochemical such as an APF receptor, such as CKAP4. In this embodiment, fractionation of the nucleus from all other cell fractions. The binding of APF to the APF-responsive protein results in the diagnostic metric in this case is the increased abundance of 30 CKAP4 (either endogenous or the stably-expressed CKAP4: translocation to the cell nucleus. YFP) relative to another marker with specificity to the The recombinant cell used in the assay of the present inven nucleus such as fibrillarin (see FIG. 4). This method would be tion may contain either an endogenous or heterologous DNA significantly more labor intensive (the biochemical fraction sequence encoding the APF-responsive protein. An endog ation would add at least one hour of human labor to the enous DNA sequence is a DNA sequence that is naturally measurement) but would also provide a quantitative measure 35 present in the genome of a cell Such that the cell naturally of CKAP4 in the nucleus. The dynamic range of this detection expresses the APF-responsive protein. A heterologous DNA method would not likely be as great as using the cell-based sequence includes a DNA sequence that does not occur natu assay if the Western blot signal were detected by enhanced rally as part of the genome in which it is present. Additionally, chemiluminescence (ECL)—captured on autofluorographic a heterologous DNA sequence includes a DNA sequence in a film. The dynamic range of ECL detection is limited by the 40 location or locations in the genome that differs from the dynamic range potential of the photographic film as well as by location(s) in which it occurs in nature. Methods for intro the length of film exposure to the signal emanating from the ducing heterologous DNA into a host cell are well known in blot. However, an approach that could provide rapid and the art and any such method may be used. sensitive detection by Western blot with a wide dynamic Capital letters indicate restriction sites described in FIG.3: range would be to label the antibodies used in the detection 45 underlined nucleotides are complimentary to the coding with radioisotope (other than tritium). The detection system sequences of their respective mRNA or cDNA. Nucleotides in this case would be an instrument such as the Phosphorim are ordered according to convention from 5' to 3'. ager (Molecular Dynamics). APF Activity Assay In the assay of the present invention, a recombinant cell is 50 CKAP4 Primers exposed to a sample which may or may not contain APF. To determine whether or not APF is present in the sample, to 5' RT PCR primer: (SEQ ID NO: 1) quantitate an amount of APF in the sample, or to measure the 5'-caccGGTACCatgcc ct cq.gc.caaacaaagg-3' biological activity of APF, for example, the expression of a 3' RT PCR primer: (SEQ ID NO: 2) reporter gene may be measured. 5'-aattcTCGAGgaccttitt cqtgaatctitctocacttitcac-3' The recombinant cell may be contacted with the sample for 55 a time sufficient to activate the receptor, for example CKAP4, YFP Primers translocate to the cell nucleus, induce transcription, and pro 5' PCR primer: (SEQ ID NO : 3) duce the reporter protein, which may or may not be secreted 5'-tatt CTCGAGatggtgagcaaggg.cgagga-3' from the cell. The time required for the assay will vary with the assay conditions and with the specific reporter protein, as 60 3 'PCR primer: (SEQ ID NO : 4) known to those in the art. Generally, the cells will be incu 5' - citcgGAATTCttacttgtacagotcgtcca-3' bated in the presence of sample for about 30 minutes to about 24 hours, after which time the reporter protein may be CKAP4 mRNA sequence: DEFINITION Homo sapiens assayed. cytoskeleton-associated protein 4 (CKAP4), mRNA. Reporter gene expression can be measured using any 65 ACCESSION NM 006825. The coding sequence, what is known method. Although it is preferable that expression of translated, nucleotides 85-1893, is underlined (SEQID NO: the reporter gene be easily detected, the reporter gene can be 5).

US 9,075,045 B2 29 30 - Continued 54 Cactggcgaa Caagttgttgt gcctgatggc Ct atcatCta citttittgcaa tgtttgttctg

6 O gtcat actgg aaaactatot ttacattacC aatgaatcct tdaaaagaat to catctgtc.

66 titatgcagag aaagatttgt tggaga.gaga gccalagagga gaagcc catc aggaagttct

72 taggcgagca gccaaggat C ttic coat cita taccaggacc atgtctggag ccatcc.gata

78 Ctgtgacaga tgccaactta taaaaccaga togctgc cat cactgct cog totgtgataa

84 atgtattittg aagatggat C at cattgtc.c atgggtgaac aattgttgttg gattittcaaa

9 O ttataagttc tttct cottt tottggctta ttctotgctic tactgcc titt tt attgcggc

96 aacagattta cagtattitta tdaaattittg gacaaatggc ctacctgata ct caa.gc.caa gttccatatt atgtttittat totttgctgc agctatotitt totgtcagot tgtc.ttctot

gtttggctat cattgttggc tagt cagcaa. aaataaatct acattagagg catt Cagaag

14 tccagtattt cga catggaa Cagataagaa tggatticago ttgggtttca gtaaaaacat

gcgacaagtt tittggtgatg agaagaagta Ctggttgcta cc cattt titt Caagttctagg

26 tgatggctgc t cott to cala cittgccttgt talaccaggat Cctgaacaag CatctactCC

32 tgcagggctg aattic cacag Ctaaaaatct cgaaaac cat cagtttic ctd caaag.c catt

38 gagagagtcC cagagccacc ttct tactga ttct cagt ct tggacggaga gcagcataaa

44 CCC aggaaaa tgcaaagctg gtatgagcaa. to ctgcatta accatggaaa atgagacitta

SO act cittcaag caagataaat toatactitta taaaagtatic aatgctgtag atggatggaa

56 gaggctt Coc acaggaaggt gccaccagtic agttgttgcct atgtc.cc titt ggctggaaat

62 gCagaatatg aattgattag ttctgttctgaa gccattgctt aaaatataac atgttittgga

68 to Caatacac acattgttac aactalacaca aattic Ctatt aaat attaala agtagttctg

74 gttt attaat Caacggggaa aaCat Cttct CCaaaaaact tggaataaat ccaaggacca

gtttittaccc aaatatatgg gtagcacagt titlatcaCata gaaactic cat taat catctg

86 attitt cogala tctgaaaatt gag act atta agat attagg attt cagaga tttcaagtica

92 cattataatg ataagcatta ttcataaaac ttgttacctt taagaaggtg galagtggcaa.

98 accatactt C tttitt tt to c totgatgtga atccago ct c agactgagtg aactgtaata

2O4. attatgaatt Cattacagag to Caggtggc Ctgcagttga agat catcaa ccatttittgc

21 O Ctcacttaat tccagcc titt tgttittctgc tggaaaataa gtgtgga cat tgaagcttga

216 gct ct caaag Cagttggctg gaatacttitt gt cagaatac gg tacatttic tattacatca

222 gaaatatatt tt cat ct citt cittgttaaat tgggaggaala tt tatgatag caattatgaa

228 gattgttitta tgacatt citt ttgtcagttt ggctttctaa aaat Ctctitt ttagattatt

234 t ct cotgttg aac at agtaa aactattgaa titt ct cittaa. gaatticcitaa tagg to aata

24 O gatttaccct c cagtgatat Citat attatt totttct cqt Ct catcaaaa. tgatgacagg

246 taalactatat titt to cittaa. acaccitatta cagttaaatt atgcaaatca ttaaataaaa.

252 at Catacaac ttittggaaag ttagttcaac atgaactaaa atggcatgct atttggaaat

258 ttagtttgag ataalactaaa. gtgtgttgat gccagaatgt to agctt cag taalatataat

264 aagct cittgt gcc ttgt atg Cactatttala aaaaagttitt ttittatttga gtc.cagtata

27 O att catgtaa atgttaacaa ttagaataat actctgtatg ctttitttgat actgattittg

276 agaatttaaa gcagattacc ttittaaaact ggaccalacta agtaattggit atttaatcaa.

282 agagaaaatg gtaataaact titt Caaaatc. tttgttaaac caaacattca acacaaaata

288 alactagaagg ccagaggata atggaataaa agat cattgc aattactitat CCtt CCtaaa.

294 aatatag titt tat attaatt gtgct tatgg aagaaacaat gt cago Caag to catttitat US 9,075,045 B2 31 32 - Continued 3 OO1 agtttgagtg caattctittgaacaatagaa at atctgcag tottt cacag atttgt atta 3 O 6 tgctgaagag titt catctga caatctgctt caagaaatct cagaaaatat gata acattt 3121 taactitt cat tittagagcac gttittggtca tttittaaaaa tacctaaagt gccagaccgg

3181 aacctatagc tactgctaga agt cittaaaa aaaccaa.ca.g cagcacagga tg tattaaga 3241 attatatgaa gtcaggtttgtttitttittitt tttitttittitt tdaaag.caca gtactgttag 3301 ctgtttttgt gga caggatt cgattaagta titccct cittg to aaactgga agct agggga 33 61 aaaagaggga tttittatcct ttact cittct agagtactgt taatgcc cct tt cocacagt 3421 ctitttatata attaaatata tdt caataca cattagaatc agatttgaaa aagttaaaac 3481 aattt cattgttgtaattgt tocctittctg ttitt catata gtgaatalacc tittaaagggit

3541 tdttttgttt tdttittgaat tataggagtt ataatetttg gagatgattg Catatotoat

36O1 tagatatgca atataaattt atctgagtga acaaagtgct aaataaatag at Ctacattt

36 61 totacatatt tatataaaat ttacctittaa gtatt tactt taaaaaattit aatggcttaa 3721 ct cqaactitg aagacacata cittcaactgt cct tattgtc cattaaactg ataattittga 3781 tttitt cittgc titttatagat tttactatat aggaatcaag atttaagaaa ttittgcatta 3841 aaaatagtgt accaatgctt catatacgitt agittatttgc tattatgtag ggaagaggat 3901 tdttatttcaaagatatatt aaagaac agt togcatctgaa tataatcatg atgcattcaa

3961 tdaagttcat atc catgaat tcact cotaa tataccctaa taaagtggitt ga

30 DHHC Protein (SEQ ID NO: 8): the primer and homology to the CKAP4 cDNA at the 3' end of the primer was used in conjunction with a 3' primer that incorporated a restriction site for the endonuclease XhoI to MAPSGPGSSARRRCRRVLYWIPVVFITLLLGWSYYAYAIOLCIVSMENTGE amplify the CKAP4 cDNA from the library. The amplified OVWCLMAYHLLFAMFVWSYWKTIFTLPMNPSKEFHLSYAEKDLLEREPRGE 35 product was isolate from an agarose gel and purified using a PCR purification kit (Qiagen). The PCR product was then AHOEVLRRAAKDLPIYTRTMSGAIRYCDRCOLIKPDRCHHCSVCDKCILKM digested using the endonucleases (X and Y) to prepare it for DHHCPWVNNCVGFSNYKFFLLFLAYSLLYCLFIAATDLOYFIKFWTNGLPD cloning into the mammalian expression vectorpcDNA3 (pur chased from Invitrogen). TOAKFHIMFLFFAAAMFSVSLSSLFGYHCWLVSKNKSTLEAFRSPVFRHGT 40 The cDNA for the Yellow Fluorescent Protein (YFP), a DKNGFSLGFSKNMROVFGDEKKYWLLPIFSSLGDGCSFPTCLVNQDPEOAS spectral mutant or color variation of the Green Fluorescent protein from the jellyfish Aequorea Victoria, may be obtained TPAGLNSTAKNLENHOFPAKPLRESOSHLLTDSOSWTESSINPGKCKAGMS from (Clontech). To subclone YFP in-frame directly to the NPALTMENET 45 carboxy-terminus of CKAP4 with no intervening linker sequence, primers complimentary to the 5' and 3' ends of the The fluorescent reporter construct CKAP4:YFP may be generated using the following methods. The cDNA for human coding sequence of monomeric YFP were made and included CKAP4 was cloned by reverse transcription polymerase XhoI and EcoRI sites respectively. chain reaction (RT-PCR) from a HeLa cell cDNA library. cDNA sequence of monomeric Aequorea victoria Yellow Primers for amplifying the cDNA were purchased from (Inte 50 Fluorescent Protein YFP DEFINITION Synthetic construct grated DNA Technologies). A 5' primer (sequence) incorpo fluorescent protein mVenus mRNA, complete cds. ACCES rating the endonuclease restriction site KpnI at the 5' end of SION DQ092360 (SEQ ID NO:9):

1 atggtgagca aggg.cgagga gctgttcacc ggggtggtgc ccatcCtggit cagctggac

61ggcgacgtaa acggccacaa gttcagcgtg tcc.ggc gagg gcgagggcga tigccaccitac 121ggcaa.gctga c cct gaagct gatctgcacc accggcaa.gc tigc.ccgtgcc Ctggc.ccacc 181 ct cqtgacca C cctgggcta cggcctgcag tgctt.cgc.cc gct accocq a cca catgaag 241 cago acgact t cittcaagtic cgc.catgc cc gaaggctacg tcc aggagcg cac catcttic 3 O1ttcaaggacg acggcaacta Caagaccc.gc gcc.gaggtga agttctgaggg cacaccCtg 361gtgaac.cgca tcgagctgaa gggcatcgac ttcaaggagg acggcaac at Cctggggcac 421 aagctggagt acaact acaa cago cacaac gtctatatica cc.gc.cgacaa goagaagaac US 9,075,045 B2 33 34 - Continued 481 ggcatcaagg cca acttcaa gatcc.gc.cac aac atcqagg acggcgg.cgt gcagctcgc.c

541 gaccact acc agcagaacac ccc catcggc gacggc.ccc.g cgacaac cac

6O1 tacctgagct accagtic caa gctgagcaaa gaccc caacg agaag.cgcga to acatggtc 661 Ctgctggagt t catgaccgc cgc.cgggat.c act ct cqgca tggacgagct gtacaagtaa ttacttgtacagct cqtcca

Protein sequence of monomeric Yellow Fluorescent Pro The invention also includes oligonucleotide, or oligonucle tein from Aequorea victoria. /protein id="AAZ65844.1 otide-like sequences Such as phosphorothioate, or peptide GI:72003.806”. The sequence of the amino acid residues is nucleic acid sequences, which possess Sufficient similarity from the amino terminus to the carboxy terminus (SEQ ID 15 with the sequences disclosed herein such that they are able to NO:10): stably hybridize to the disclosed sequences, or their comple ments. Such sequences may be intended as antisense regula tors of gene expression, or for the selective amplification or MWSKGEELFTGVWPILWELDGDWNGHKFSWSGEGEGDATYGKLT extension of adjoining sequences, for instance by PCR using LKLICTTGKLPVPWPTLVTTLGYGLOCFARYPDHMKOHDFFKSAMPEGYVO a given annealing temperature, as would be determined by someone skilled in the art. In addition to the sequences dis ERTIFFKDDGNYKTRAEWKFEGDTLWNRIELKGIDFKEDGNILGHKLEYNY closed here, related sequences in other organisms, or homologs, will be readily identified by hybridization using NSHNVYITADKOKNGIKANEKIRHNIEDGGVOLADHYOONTPIGDGPWLLP the present sequences. Similar techniques will also apply to DNHYLSYOSKLSKDPNEKRDHMVLLEFWTAAGITLGMDELYK 25 the identification of mutant alleles, polymorphisms, dele tions, insertions, and so forth, in genomic and cDNA One aspect of the present invention is the polynucleotide sequences. Whole or partial sequences referred to above may sequences essentially as set forth above, the complement of also be identified and isolated using techniques that involve these sequences, the RNA versions of both DNA strands and annealing of short oligonucleotides to complementary the information otherwise contained within the linear 30 sequences, such as those as might be present in the genomic sequence of these polynucleotide sequences, and fragments DNA of a particular organism, or in genomic or cDNA, thereof. The polynucleotide encoding CKAP4, APF, and/or including expression cDNA, libraries. Thus, PCR is used to DHHC2 is exemplified as set forth above. In the case of obtain DNA sequences homologous to, and which lie nucleic acid segments, sequences for use with the present between, two primers, usually between 15 to 30 nucleotides invention are those that have greater than about 50 to 60% 35 which have annealing temperatures typically between 60-80 homology with any portion of the polynucleotide sequences degrees Celsius may be substantially purified. described herein, sequences that have between about 61% It will be understood that this invention is not limited to the and about 70%; sequences that have between about 71 and particular nucleic acid sequences presented herein. Recom about 80%; or between about 81% and about 90%; or between binant vectors, including for example plasmids, phage, 91% and about 99%; or which contain nucleotides that are 40 viruses, and other sequences, and isolated DNA or RNA identical, functionality equivalent, or functionally irrelevant, segments may therefore variously include the CKAP4, APF with respect to the nucleotides set forth above are considered and/or DHHC2 gene sequences or their complements, and to be essentially similar. Also encompassed within the present coding regions, as well as those that may bear selected alter invention are nucleic acids that encode polypeptides that are ations or modifications that nevertheless include CKAP4, at least 40% identical or similar to the amino acid sequences 45 APF and/or DHHC2 segments or may encode biologically or as shown. experimentally relevant amino acid sequences. Such The invention also encompasses other nucleic acids or sequences may be created by the application of recombinant nucleic acid like molecules that are sufficient in any regard to DNA technology, where changes are engineered based on the mimic, substitute for, or interfere with the CKAP4 and/or consideration of the nucleotides or amino acids being APF polynucleotide sequences, as exemplified as set forth 50 exchanged, deleted, inserted, fused, or otherwise modified. above, or fragments thereof. It will also be understood that the Proteins and Polypeptides nucleic acid and amino acid sequences may include addi One aspect of the invention is the protein, polypeptide, tional residues, such as additional 5'- or 3'-sequences, and yet oligopeptide, or amino acid sequences or fragments thereof, still be essentially as set forth in one of the sequences dis of CKAP4, APF and/or DHHC2, essentially as set forth closed herein, so long as the sequence meets the criteria set 55 above. The CKAP4, APF and/or DHHC2 polypeptide is forth, including the maintenance of functionality, or for the exemplified above. Sequences that have greater than about purpose of engineering altered functionality with respect to 40-50% homology with any portion of the amino acid CKAP4, DHHC2 and/or APF. sequences described herein, sequences that have between Included within the invention are DNA or RNA segments about 51% and about 60%; sequences that have between including oligonucleotides, polynucleotides and fragments 60 about 61% and about 70% sequences that have between about thereof, including DNA or RNA or nucleic acid-like 70 and about 80%; or between about 81% and about 90%; or sequences of genomic or synthetic origin, single or double between 91% and about 99%; or those that contain amino Stranded. The invention includes nucleic acid molecules, or acids that are identical, functionally equivalent, or function nucleic acid-like molecules that are able to hybridize to the ally irrelevant, for instance those specified by conservative, sequences as set forth above, under stringent or under per 65 evolutionarily conserved, and degenerate Substitutions, with missive hybridization conditions, or to the complement of respect to the amino acid sequences presented above are said sequences. included. The invention thus applies to CKAP4, APF and/or US 9,075,045 B2 35 36 DHHC2 polypeptide sequences, or fragments thereof, and those proficient in the art. Cells for such production are nucleic acids which encode such polypeptides. Such as those known to include yeast and other fungi, Drosophila and Sf9 of other species. Reference is particularly, but not exclusively, cells, cells of other higher eukaryotic organisms such as made to the conserved regions of CKAP4, APF and/or HeLa, COS, CHO and others, as well as plant cells. Similarly, DHHC2, in contrast to similarity throughout the entire length. 5 expression could be achieved in prokaryotic or eukaryotic The invention thus encompasses amino acid sequences, or extracts that are able to translate RNAs into proteins, such as amino acid-like molecules, that are sufficient in any regard to rabbit reticulocyte lysates. mimic, substitute for, or interfere with the CKAP4, APF Vectors and/or DHHC2 amino acid sequences, or fragments thereof. Prokaryote- and/or eukaryote-based systems can be The invention encompasses CKAP4, APF and/or DHHC2 10 employed for use with the present invention to produce amino acid sequences that have been altered in any form, nucleic acid sequences, or their cognate polypeptides, pro either through the use of recombinant engineering, or through teins and peptides. Many such systems are commercially and post-translational or chemical modifications, including those widely available. that may be produced by natural, biological, artificial, or The insect cell/baculovirus system can produce a high level chemical methods. Naturally, it will be understood that this 15 of protein expression of a heterologous nucleic acid segment, invention is not limited to the particular amino acid sequences such as described in U.S. Pat. Nos. 5,871,986, 4,879,236, presented herein. Altered amino acid sequences include those both herein incorporated by reference, and which can be which have been created by the application of recombinant bought, for example, under the name MAXBAC(R). 2.0 from technology Such that specific residues, regions, or domains INVITROGENCR) and BACPACKCR) BACULOVIRUS have been altered, and which may be functionally identical, or EXPRESSION SYSTEM FROM CLONTECH. which may possess unique biological or experimental prop Vectors may be of bacterial origin, which may comprise a erties with regards to function or interactions with natural and promoter of a bacteriophage such as phage or T7 which is artificial ligands. capable of functioning in the bacteria. In one of the most For instance Such modifications may confer longer or widely used expression systems, the nucleic acid encoding shorter half-life, reduced or increased sensitivity to ligands 25 the CKAP4, APF and/or DHHC2 may be transcribed from the that modify function, ability to detect or purify polypeptides, vector by T7 RNA polymerase (Studier et al. Methods in solubility, and so forth. Alternatively, Such sequences may be Enzymol. 185: 60-89, 1990). In the E. coli BL21 (DE3) host shorter oligopeptides that possess an antigenic determinant, strain, used in conjunction with plT vectors, the T7 RNA or property that interferes, or competes, with the function of polymerase is produced from the 1-lysogen DE3 in the host a larger polypeptide, and those that affect interactions 30 bacterium, and its expression is under the control of the IPTG between CKAP4, APF and/or DHHC2 other proteins, other inducible lac UV5 promoter. This system has been employed nucleic acid regions, and other proteins. Such sequences may successfully for over-production of many proteins. Alterna be created by the application of the nucleotides oramino acids tively, the polymerase gene may be introduced on a lambda being exchanged, deleted, inserted, fused, or otherwise modi phage by infection with an int-phage Such as the CE6 phage, fied. Likewise, the current invention within, the sequences 35 which is commercially available (Novagen, Madison, USA). that may be naturally present as extensions of or insertions Other vectors include vectors containing the lambda PL pro within, the sequences disclosed herein, including alternative moter such as PLEXOR (Invitrogen, NL), vectors containing or longer N- and C-terminal sequences, or alternatively the trc promoters such as pTrcH is Xpress(R (Invitrogen), or spliced protein isoforms. pTrc99 (Pharmacia Biotech, SE), or vectors containing the Production and purification of polypeptides may be 40 tac promoter such as pKK223-3 (Pharmacia Biotech), or achieved in any of a variety of expression systems known to PMAL (New England Biolabs, MA, USA). those skilled in the art, including recombinant DNA tech One of skill in the art will understand that cloning also niques, genetic recombination, and chemical synthesis. For requires the step of transforming a host cell with a recombi instance, expression in prokaryotic cells may be achieved by nant nucleic acid molecule. A host cell is “transformed by a placing protein coding nucleic sequences downstream of a 45 nucleic acid when the nucleic acid is translocated into the cell promoter, such as T7, T3, lacI, lacz, tip, or other cellular, from the extracellular environment. Any method of transfer viral, or artificially modified promoters including those that ring a nucleic acid into the cell may be used; the term, unless may be inducible by IPTG, tetracycline, maltose, and so forth. otherwise indicated herein, does not imply any particular Such promoters are often provided for in commercially avail method of delivering a nucleic acid into a cell, nor that any able recombinant DNA vectors such as pRSET ABC, pBlue 50 particular cell type is the subject of transfer. For example, script, pKK223-3, and others, or are easily constructed to bacterial host cells, such as E. coli HB101, can be trans achieve Such a purpose, and often include the presence of formed by electroporation using any commercially-available multiple cloning sites (MCS) to facilitate typically contain electroporation apparatus known in the art, such as a efficient ribosome binding sites, and in some cases transcrip GenePulser apparatus (Bio-Rad, Hercules, Calif.). In one tion termination signals. 55 embodiment, mammaliancells, such as BHK-21 cells or Vero Peptides, oligopeptides and polypeptides may also be pro cells (ATCC CCL-81), are transformed with a recombinant duced by chemical synthesis, for instance Solid phase tech plasmid containing a cloned cDNA by the method of “trans niques, either manually or under automated control Such as fection.” The term “transfection” refers to the transfer of Applied Biosystems 431 peptide synthesizer (PerkinElmer). genetic material into a eukaryotic cell. Such as a mammalian After synthesis, such molecules are often further purified by 60 cell, from the external environment of the cell. preparative high performance liquid chromatography. Thus, One of skill in the art will appreciate the variety of methods the invention provides methods for the production of epitopes of transfection that are available in the art. Such methods for antibody production, or the production of small molecules include the nucleic acid/CaPO4 co-precipitation method, the that enhance or interfere with a specific function or interac diethylaminoethyl (DEAE)-dextran method, the polybrene tion of the CKAP4, APF and/or DHHC2 polypeptides. 65 method, the cationic liposome method (“lipofection'), the Methods to produce and purify said polypeptides in electroporation method, the microinjection method, and the eukaryotic systems are widely available and understood by microparticle bombardment method. A description of trans US 9,075,045 B2 37 38 fection methods can be found in M. A. Aitken et al., Molecu Plasmid Vectors lar Biomethods Handbook, Chapter 20, p. 235-250. Any plasmid vector that allows expression of a differen According to another embodiment of the instant invention, tially expressed coding sequence of the inventionina selected in vitro transcription is carried out on a recombinant plasmid host cell type is acceptable for use according to the invention. carrying a cloned cDNA of the invention, under the control of 5 A plasmid vector useful in the invention may have any or all an expressible promoter (i.e., a promoter which is effectively of the above-noted characteristics of vectors useful according enabled or activated in vitro in the presence of corresponding to the invention. Plasmid vectors useful according to the transcription factors and RNA polymerase). The transcription invention include, but are not limited to the following process generates a fully-infectious mRNA transcript that can examples: Bacterial pOE70, pCRE60, pGE-9 (Qiagen) pBs, 10 phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, be used to transfect (i.e., infect) a cell host, such as BHK-21 pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, (hamster kidney cells) or Vero cells. In one embodiment, the pKK223-3, pKK233-3, pIDR540, and pRIT5 (Pharmacia): cDNA is operably linked with the bacteriophage transcrip Eukaryotic pWLineo, pSV2cat, pCG44, pXT1, pSG (Strat tional promoter, T7; to enable the in vitro transcription of the agene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). How cDNA using bacteriophage T7 DNA-dependent RNA poly 15 ever, any other plasmid or vector may be used as long as it is merase. One of ordinary skill in the art will appreciate that any replicable and viable in the host. suitable promoter, such as, for example, SP6, T3, any bacte Bacteriophage Vectors. rial, viral, phage, or eukaryotic promoter, for controlling the There are a number of well known bacteriophage-derived transcription of for example, the CKAP4, APF and/or vectors useful according to the invention. Foremost among DHHC2 gene, or fragment thereof, and for controlling the these are the lambda-based vectors, such as Lambda Zap II or expression of a nucleotide sequence encoding a reporter is Lambda-Zap. Express vectors (Stratagene) that allow induc contemplated by the present invention. It will be appreciated ible expression of the polypeptide encoded by the insert. that the promoter is typically selected from promoters which Others include filamentous bacteriophage such as the M13 are functional in mammalian cells Susceptible to infection by based family of vectors. the CKAP4, APF and/or DHHC2 gene, or fragment thereof, 25 Viral Vectors. encoding sequences of the invention, although prokaryotic or A number of different viral vectors are useful according to phage promoters and promoters functional in other eukary the invention, and any viral vector that permits the introduc otic cells may be used. The promoter is typically derived from tion and expression of one or more of the differentially promoter sequences of viral or eukaryotic genes. For expressed polynucleotides of the invention in cells is accept example, it may be a promoter derived from the genome of a 30 able for use in the methods of the invention. Viral vectors that cell in which expression or transcription of, for example, the can be used to deliver foreign nucleic acid into cells include CKAP4 and/or APF gene, or fragment thereof, encoding but are not limited to retroviral vectors, adenoviral vectors, sequence or construct is to occur. adeno-associated viral vectors, herpesviral vectors, and Sem With respect to eukaryotic promoters, they may be promot liki forest viral (alphaviral) vectors. Defective retroviruses 35 are well characterized for use in gene transfer (for a review ers that function in a ubiquitous manner (Such as promoters of see Miller, A. D. (1990) Blood 76:271). Protocols for produc C.-actin, B-actin, tubulin) or, alternatively, a tissue-specific ing recombinant retroviruses and for infecting cells in vitro or manner (such as promoters of the genes for pyruvate kinase). in vivo with such viruses can be found in Current Protocols in Tissue-specific or cell-specific promoters specific for lym Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Pub phocytes, dendritic cells, skin, brain cells and epithelial cells, 40 lishing Associates, (1989), Sections 9.10-9.14, and other for example the CD2, CD11c, keratin 14, Wnt-1 and Rhodop standard laboratory manuals. sin promoters, respectively. For example, the epithelial cell In addition to retroviral vectors, Adenovirus can be promoter SPC is used. They may also be promoters that manipulated Such that it encodes and expresses a gene prod respond to specific stimuli, for example promoters that bind uct of interest but is inactivated in terms of its ability to steroid hormone receptors. Viral promoters may also be used, 45 replicate in a normal lytic viral life cycle (see for example for example the Moloney murine leukaemia virus long termi Berkner et al., 1988, BioTechniques 6:616; Rosenfeld et al., nal repeat (MMLV LTR) promoter, the rous sarcoma virus 1991, Science 252:431-434; and Rosenfeld et al., 1992, Cell (RSV) LTR promoter, the human cytomegalovirus (CMV) IE 68: 143-155). Suitable adenoviral vectors derived from the promoter, or SV40 promoter. adenovirus strain Ad type 5 d1324 or other strains of aden It may also be advantageous for the promoters to be induc 50 ovirus (e.g., Ad2. Ad3, Ad7 etc.) are well known to those ible so that the levels of expression of, for example, the skilled in the art. Adeno-associated virus (AAV) is a naturally CKAP4, APF and/or DHHC2 gene, or fragment thereof occurring defective virus that requires another virus, Such as encoding sequence can be regulated during the life-time of an adenovirus or a herpes virus, as a helper virus for efficient the cell. Inducible means that the levels of expression replication and a productive life cycle. (For a review see obtained using the promoter can be regulated. 55 Muzyczka et al., 1992, Curr. Topics in Micro. and Immunol. In addition, any of these promoters may be modified by the 158:97-129). An AAV vector such as that described in Tra addition of further regulatory sequences, for example schin et al. (1985, Mol. Cell. Biol. 5:3251-3260) can be used enhancer sequences. Chimeric promoters may also be used to introduce nucleic acid into cells. A variety of nucleic acids comprising sequence elements from two or more different have been introduced into different cell types using AAV promoters described above. It will be appreciated that the 60 vectors (see, for example, Hermonat et al., 1984, Proc. Natl. Sources of promoter sequences, which typically can be Acad. Sci. USA 81: 6466-6470; and Traschin et al., 1985, retrieved using recombinant techniques from different clon Mol. Cell. Biol. 4: 2072-2081). ing vectors and plasmids, etc., can be obtained from commer Introduction of Vectors to Host Cells cial sources, such as, NEW ENGLAND BIOLABS, INC. Vectors useful in the present invention may be introduced (MA), PROMEGA CORPORATION (WI), or BD BIO 65 to selected host cells by any of a number of suitable methods SCIENCES (CA), or from the laboratories of academic known to those skilled in the art. For example, vector con research groups upon request. structs may be introduced to appropriate bacterial cells by US 9,075,045 B2 39 40 infection, in the case of E. coli bacteriophage vector particles vectors would include those with inserts present in either such as lambda or M13, or by any of a number of transfor forward or reverse orientations. mation methods for plasmid vectors or for bacteriophage Many of the vectors and hosts have specific features that DNA. For example, standard calcium-chloride-mediated facilitate expression or Subsequent purification. For instance bacterial transformation is still commonly used to introduce 5 DNA sequences to be expressed as proteins often appear as naked DNA to bacteria (Sambrook et al., 1989, Molecular fusion with unrelated sequences that encode polyhistidine Cloning, A Laboratory Manual, Cold Spring Harbor Labora tags, or HA, FLAG, myc and other epitope tags for immu tory Press, Cold Spring Harbor, N.Y.), but electroporation nochemical purification and detection, or phosphorylation may also be used (Ausubel et al., 1988, Current Protocols in sites, or protease recognition sites, or additional protein Molecular Biology, (John Wiley & Sons, Inc., NY, N.Y.)). 10 domains such as glutathione S-transferase (GST), maltose For the introduction of vector constructs to yeast or other binding protein (MBP), and so forth which facilitate purifi fungal cells, chemical transformation methods are generally cation. Vectors may also be designed which contain elements used (e.g. as described by Rose et al., 1990, Methods in Yeast for polyadenylation, splicing and termination, such that Genetics, Cold Spring Harbor Laboratory Press, Cold Spring incorporation of naturally occurring genomic DNA Harbor, N.Y.). For transformation of S. cerevisiae, for 15 sequences that contain introns and exons can be produced and example, the cells are treated with lithium acetate to achieve processed, or Such that unrelated introns and other regulatory transformation efficiencies of approximately 104 colony signals require RNA processing prior to production of forming units (transformed cells)/.mu.g. of DNA. Trans mature, translatable RNAs. Proteins produced in the systems formed cells are then isolated on selective media appropriate described above could be subject to a variety of post-transla to the selectable marker used. Alternatively, or in addition, tional modifications, such as glycosylation, phosphorylation, plates or filters lifted from plates may be scanned for GFP nonspecific or specific proteolysis or processing. fluorescence to identify transformed clones. Purification of CKAP4, APF and/or DHHC2, or variants For the introduction of vectors comprising differentially produced as described above can be achieved by any of sev expressed sequences to mammalian cells, the method used eral widely available methods. Cells may be subject to freeze will depend upon the form of the vector. Plasmid vectors may 25 thaw cycles or Sonication to achieve disruption, or may be be introduced by any of a number of transfection methods, fractionated into subcellular components prior to further puri including, for example, lipid-mediated transfection ("lipofec fication. Purification may be achieved by one or more tech tion'), DEAE-dextran-mediated transfection, electropora niques such as precipitation with salts or organic solvents, ion tion or calcium phosphate precipitation. These methods are exchange, hydrophobic interaction, HPLC and FPLC chro detailed, for example, in Current Protocols in Molecular Biol 30 matographic techniques. Affinity chromatographic tech ogy (Ausubel et al., 1988, John Wiley & Sons, Inc., NY, N.Y.). niques could include the use of polyclonal or monoclonal Lipofection reagents and methods suitable for transient antibodies raised against the expressed polypeptide, or anti transfection of a wide variety of transformed and non-trans bodies raised against or available for an epitopic tag. Such as formed or primary cells are widely available, making lipofec HA or FLAG. Similarly, purification can be aided by affinity tion an attractive method of introducing constructs to eukary 35 chromatography using fusions to the desired proteins such as otic, and particularly mammalian cells in culture. For GSH-affinity resin, maltose affinity resin, carbohydrate (lec example, LipofectamineTM (Life Technologies) or Lipo tin) affinity resin or, in a one embodiment, Ni-affinity resin, TaxiTM (Stratagene) kits are available. Other companies offer and so forth. In some instances purification is achieved in the ing reagents and methods for lipofection include BioRad presence of denaturing agents such as urea or guanidine, and Laboratories, CLONTECH, Glen Research, InVitrogen, JBL 40 Subsequent dialysis techniques may be required to restore Scientific, MBI Fermentas, PanVera, Promega, Quantum functionality, if desired. Biotechnologies, Sigma-Aldrich, and Wako Chemicals USA. Any method of in vitro transcription known to one of Following transfection with a vector of the invention, eukary ordinary skill in the art is contemplated by the instant inven otic (e.g., human) cells successfully incorporating the con tion. It will be understood that the method of in vitro tran struct (intra- or extrachromosomally) may be selected, as 45 Scription of a DNA sequence relies on the operable linkage to noted above, by either treatment of the transfected population an appropriate promoter and that the cognate RNA poly with a selection agent, Such as an antibiotic whose resistance merase is used to direct transcription of the DNA starting at gene is encoded by the vector, or by direct screening using, for the promoter sequence. It will be further appreciated that the example, FACS of the cell population or fluorescence scan RNA polymerase and promoter can be of bacterial, eukary ning of adherent cultures. Frequently, both types of screening 50 otic, or viral (including bacteriophage) origin. Bacterioph may be used, whereina negative selection is used to enrich for age-RNA polymerases are very robust, and the availability of cells taking up the construct and FACS or fluorescence scan purified recombinant proteins facilitates the generation of ning is used to further enrich for cells expressing differen large quantities of RNA from cloned cDNA sequences. In tially expressed polynucleotides or to identify specific clones contrast, eukaryotic in vitro transcription systems yield rela of cells, respectively. For example, a negative selection with 55 tively small quantities of RNA. Bacteriophage-RNA poly the neomycin analog G418 (Life Technologies, Inc.) may be merases, such as from bacteriophages SP6, T7, and T3, are used to identify cells that have received the vector, and fluo especially suitable for the generation of RNA from DNA rescence Scanning may be used to identify those cells or sequences cloned downstream of their specific promoters clones of cells that express the vector construct to the greatest because, first, their promoters are Small and easily incorpo eXtent. 60 rated into plasmid vectors and second, the polymerases are The invention also relates to cells which contain such quite specific for their cognate promoters, which results in recombinant constructs, where the host cell refers to mam very little incorrect transcriptional initiation from DNA tem malian, plant, yeast, insect, or other eukaryotic cells, or to plates. Any suitable promoter, however, is contemplated by prokaryotic, or archae, and vectors that are designed for a the instant invention, including, for example, bacterial, given host. Promoter-vector combinations could be chosenby 65 phage, viral, and eukaryotic promoters. Strong termination a person skilled in these arts. In some cases, the desired sequences are not available for these polymerases so that outcome may not be protein, but RNA, and recombinant DNA templates can be linearized with a restriction enzyme 3 US 9,075,045 B2 41 42 to the desired end of the RNA transcript and the polymerase included within a construct that also includes a heterologous is forced to stop at this point—a process referred to as "run DNA sequence encoding a APF-responsive protein. In an off transcription. A full description of in vitro transcription exemplary embodiment, the recombinant assay cell stably can be found in M. A. Aitken et al., Molecular Biomethods expresses both the APF-responsive protein and the reporter Handbook, Chapter 26, p. 327-334 and Sambrook, J. and D. gene. W. Russell, Molecular Cloning: A Laboratory Manual. Third An appropriate host can be determined by one of skill in the Edition (2001). art based on the vector backbone and the desired result. A The invention provides a dual luciferase reporter system plasmid or cosmid, for example, can be introduced into a for measuring recoding efficiencies in Vivo or in vitro from a prokaryote host cell for replication of many vectors. Bacterial single construct see U.S. Pat. No. 6,143,502 (Grentzmann et 10 cells used as host cells for vector replication and/or expres al.). For example, the firefly luciferase gene (fluc) has been sion include DH5O, JM109, and KC8, as well as a number of cloned behind the renilla luciferase gene (rluc) into an altered commercially available bacterial hosts such as SURE(R). vector pRL-SV40 vector (Promega Corp., Madison, W is: Competent Cells and SOLOPACKR) Gold Cells (STRAT catalog no. TB239). Other reporter genes may also be used, AGENER), La Jolla). Alternatively, bacterial cells such as E. for example, green fluorescent protein, and variants thereof. 15 coli LE392 could be used as host cells for phage viruses. Expression features for initiation and termination of tran Examples of eukaryotic host cells for replication and/or Scription and translation, as well as the nature of the two expression of a vector include HeLa, NIH3T3, Jurkat, 293, reporter genes (short enough to be efficiently synthesized in Cos, CHO, Saos, and PC12, etc. Many host cells from various an in vitro translation system), allow application of the same cell types and organisms are available and would be known to reporter construct for in vivo and in vitro applications. one of skill in the art. Similarly, a viral vector may be used in Between the 5' reporter (rluc) and the 3' reporter (fluc) two conjunction with either a eukaryotic or prokaryotic host cell, alternative polylinkers have been inserted, yielding p2luc and particularly one that is permissive for replication or expres p2luci. The p2luc polylinker has restriction sites for digestion sion of the vector. with SalI, BamHI, and SacI, whereas the p2luci polylinker Reporter Genes has restriction sites for digestion with SalI. Apal, BglII, 25 The DNA encoding the reporter gene can be included Eco47 ul, BamHI, SmaI, and SacI. The assay using these within a DNA construct which further includes a transcrip reporter plasmids combines rapidity of the reactions with tional control sequence. A transcriptional control sequence very low background levels and provides a powerful assay. In can include regulatory elements such as promoter sequences, vitro experiments can be performed in 96-well microtiter enhancer sequences, repressor sequences and the like. plates, and in vivo experiments can be performed in 6-well 30 According to the present invention, the activity of the tran culture dishes. This makes the dual-luciferase assay suitable Scriptional control sequence can be affected by the binding of for high throughput screening approaches. APF to a cell surface receptor, such as CKAP4. For example, Host Cells the transcriptional control element is a APF/CKAP4-respon As used herein, the terms “cell,” “cell line, and “cell sive promoter, which in the presence of APF/CKAP4, modu culture' may be used interchangeably. All of these term also 35 lates expression of the reporter. For example, the APF/ include their progeny, which is any and all Subsequent gen CKAP4-responsive promoter induces activity of the erations. It is understood that all progeny may not be identical promoter in the presence of APF/CKAP4, resulting in expres due to deliberate or inadvertent mutations. In the context of sion of the reporter protein. expressing a heterologous nucleic acid sequence, "host cell” A promoter is a region of DNA upstream from the reporter refers to a prokaryotic or eukaryotic cell, and it includes any 40 gene with respect to the direction of transcription of the transformable organisms that is capable of replicating a vec reporter gene and a transcription initiation site. It includes the tor and/or expressing a heterologous gene encoded by a vec RNA polymerase binding and transcription initiation sites tor. A host cell can, and has been, used as a recipient for and other regions such as repressor or activator protein bind vectors. A host cell may be “transfected' or “transformed.” ing sites. which refers to a process by which exogenous nucleic acid is 45 The transcriptional control sequence is operatively linked transferred or introduced into the host cell. A transformed cell to the DNA sequence encoding the reporter gene. Typically, includes the primary Subject cell and its progeny. the DNA sequences are included in a vector Such as a plasmid, A suitable recombinant cell for use in the present invention bacteriophage or yeast chromosome. The vector can also includes a protein that is responsive to APF and a reporter include other gene sequences such as a DNA sequence encod gene whose expression is modulated by the binding of APF to 50 ing the APF-responsive protein, resistance genes, enhancers the APF-responsive protein, such as, for example, CKAP4. or other regulatory elements such as terminators, polyadeny "Modulated can include up-regulation or down-regulation. lation sequences or nucleic acid sequences encoding signal A heterologous DNA sequence encoding the reporter gene peptides to direct the encoded protein to the cell surface. can be introduced into the recombinant cell by transient trans Fluorescent Protein Variants fection or by stable transfection of a vector containing the 55 The present invention also provides polynucleotides DNA sequence. Numerous methods are know for introducing encoding fluorescent protein variants as the reporter, where foreign DNA into a host cell. the protein can be a dimeric fluorescent protein, a tandem In one embodiment, the host cell contains an endogenous dimeric fluorescent protein, a monomeric protein, or a fusion DNA sequence encoding a APF-responsive protein. In this protein comprising a fluorescent protein operatively linked to embodiment, a DNA sequence encoding the reporter gene is 60 one or more polypeptides of interest. In the case of the tandem introduced into the cell. In another embodiment, the host cell dimer the entire dimer may be encoded by one polynucleotide may or may not contain an endogenous DNA sequence molecule. If the linker is a non-peptide linker, the two sub encoding a APF-responsive protein. In this embodiment, a units will be encoded by separate polynucleotide molecules, heterologous DNA sequence encoding the reporter gene can produced separately, and Subsequently linked by methods be co-transfected with another heterologous DNA sequence 65 known in the art. encoding a APF responsive protein. Alternatively, the heter The invention further concerns vectors containing Such ologous DNA sequence encoding the reporter gene can be polynucleotides, and host cell containing a polynucleotide or US 9,075,045 B2 43 44 vector. Also provided is a recombinant nucleic acid molecule, PCR of cDNA of the Discosoma coral, or obtained from the which includes at least one polynucleotide encoding a fluo commercially available Dsked2 or HCRed1 (CLONTECH). rescent protein variant operatively linked to one or more other PCR methods are well known and routine in the art (see, for polynucleotides. The one or more other polynucleotides can example, U.S. Pat. No. 4,683, 195; Mullis et al., Cold Spring be, for example, a transcription regulatory element such as a Harbor Symp. Quant. Biol. 51:263, 1987: Erlich, ed., “PCR promoter or polyadenylation signal sequence, or a translation Technology” (Stockton Press, NY. 1989)). A variant form of regulatory element Such as a ribosome binding site. Such a the fluorescent protein then can be made by site-specific recombinant nucleic acid molecule can be contained in a mutagenesis of the polynucleotide encoding the fluorescent vector, which can be an expression vector, and the nucleic protein. Similarly, a tandem dimer fluorescent protein can be acid molecule or the vector can be contained in a host cell. 10 expressed from a polynucleotide prepared by PCR or The vector generally contains elements required for repli obtained otherwise, using primers that can encode, for cation in a prokaryotic or eukaryotic host system or both, as example, a peptide linker, which operatively links a first desired. Such vectors, which include plasmid vectors and monomer and at least a second monomer of a fluorescent viral vectors such as bacteriophage, baculovirus, retrovirus, protein. lentivirus, adenovirus, vaccinia virus, Semliki forest virus and 15 The construction of expression vectors and the expression adeno-associated virus vectors, are well known and can be of a polynucleotide in transfected cells involves the use of purchased from a commercial source (Promega, Madison molecular cloning techniques also well known in the art (see Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Sambrook et al. In “Molecular Cloning: A Laboratory Md.) or can be constructed by one skilled in the art (see, for Manual” (Cold Spring Harbor Laboratory Press 1989); "Cur example, Meth. Enzymol. Vol. 185, Goeddel, ed. (Academic rent Protocols in Molecular Biology’ (eds., Ausubel et al.: Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994: Greene Publishing Associates, Inc., and John Wiley & Sons, Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum Inc. 1990 and supplements). Expression vectors contain et al., J. Clin. Invest. 92:381-387, 1993; each of which is expression control sequences operatively linked to a poly incorporated herein by reference). nucleotide sequence of interest, for example, that encodes a A vector for containing a polynucleotide encoding a fluo 25 fluorescent protein variant, as indicated above. The expres rescent protein variant can be a cloning vector or an expres sion vector can be adapted for function in prokaryotes or sion vector, and can be a plasmid vector, viral vector, and the eukaryotes by inclusion of appropriate promoters, replication like. Generally, the vector contains a selectable marker inde sequences, markers, and the like. An expression vector can be pendent of that encoded by a polynucleotide of the invention, transfected into a recombinant host cell for expression of a and further can contain transcription or translation regulatory 30 fluorescent protein variant, and host cells can be selected, for elements, including a promoter sequence, which can provide example, for high levels of expression in order to obtain a tissue specific expression of a polynucleotide operatively large amount of isolated protein. A host cell can be main linked thereto, which can, but need not, be the polynucleotide tained in cell culture, or can be a cell in vivo in an organism. encoding the fluorescent protein variant, for example, a tan A fluorescent protein variant can be produced by expression dem dimer fluorescent protein, thus providing a means to 35 from a polynucleotide encoding the protein in a host cell Such select a particular cell type from among a mixed population of as E. coli. Aequorea GFP-related fluorescent proteins, for cells containing the introduced vector and recombinant example, are best expressed by cells cultured between about nucleic acid molecule contained therein. 15° C. and 30°C., although higher temperatures such as 37 Where the vector is a viral vector, it can be selected based C. can be used. After synthesis, the fluorescent proteins are on its ability to infect one or few specific cell types with 40 stable at higher temperatures and can be used in assays at Such relatively high efficiency. For example, the viral vector also temperatures. can be derived from a virus that infects particular cells of an An expressed fluorescent protein variant, which can be a organism of interest, for example, Vertebrate host cells Such tandem dimer fluorescent protein or a non-oligomerizing as mammalian host cells. Viral vectors have been developed monomer, can be operatively linked to a first polypeptide of for use in particular host systems, particularly mammalian 45 interest, further can be linked to a second polypeptide of systems and include, for example, retroviral vectors, other interest, for example, a peptide tag, which can be used to lentivirus vectors such as those based on the human immu facilitate isolation of the fluorescent protein variant, includ nodeficiency virus (HIV), adenovirus vectors, adeno-associ ing any other polypeptides linked thereto. For example, a ated virus vectors, herpesvirus vectors, vaccinia virus vectors, polyhistidine tag containing, for example, six histidine resi and the like (see Miller and Rosman, BioTechniques 7: 980 50 dues, can be incorporated at the N-terminus or C-terminus of 990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998: the fluorescent protein variant, which then can be isolated in Verma and Somia, Nature 389:239-242, 1997: Wilson, New a single step using nickel-chelate chromatography. Addi Engl. J. Med. 334:1185-1187 (1996), each of which is incor tional peptide tags, including a c-myc peptide, a FLAG porated herein by reference). epitope, or any ligand (or cognate receptor), including any Recombinant production of a fluorescent protein variant, 55 peptide epitope (or antibody, or antigen binding fragment which can be a component of a fusion protein, involves thereof, that specifically binds the epitope are well known in expressing a polypeptide encoded by a polynucleotide. A the art and similarly can be used. (see, for example, Hoppet polynucleotide encoding the fluorescent protein variant is a al., Biotechnology 6:1204 (1988): U.S. Pat. No. 5,011,912, useful starting material. Polynucleotides encoding fluores each of which is incorporated herein by reference). cent protein are disclosed herein or otherwise known in the 60 Uses of Fluorescent Protein Variants art, and can be obtained using routine methods, then can be A fluorescent protein variant of the invention is useful in modified such that the encoded fluorescent protein lacks a any method that employs a fluorescent protein. Thus, the propensity to oligomerize. For example, a polynucleotide fluorescent protein variants, including the monomeric, encoding a GFP can be isolated by PCR of cDNA from A. dimeric, and tandem dimer fluorescent proteins, are useful as victoria using primers based on the DNA sequence of Aequo 65 fluorescent markers in the many ways fluorescent markers rea GFP. A polynucleotide encoding the red fluorescent pro already are used, including, for example, coupling fluorescent tein from Discosoma (Dsked) can be similarly isolated by protein variants to antibodies, polynucleotides or other recep US 9,075,045 B2 45 46 tors for use in detection assays Such as immunoassays or perature while it is being scanned, and can have a multi-axis hybridization assays, or to track the movement of proteins in translation stage, which moves a microtiter plate holding a cells. For intracellular tracking studies, a first (or other) poly plurality of samples in order to position different wells to be nucleotide encoding the fluorescent protein variant is fused to exposed. The multi-axis translation stage, temperature con a second (or other) polynucleotide encoding a protein of 5 troller, auto-focusing feature, and electronics associated with interest and the construct, if desired, can be inserted into an imaging and data collection can be managed by an appropri expression vector. Upon expression inside the cell, the protein ately programmed digital computer, which also can transform of interest can be localized based on fluorescence, without the data collected during the assay into another format for concern that localization of the protein is an artifact caused by presentation. This process can be miniaturized and automated oligomerization of the fluorescent protein component of the 10 to enable screening many thousands of compounds in a high fusion protein. In one embodiment of this method, two pro throughput format. These and other methods of performing teins of interest independently are fused with two fluorescent assays on fluorescent materials are well known in the art (see, protein variants that have different fluorescent characteristics. for example, Lakowicz, "Principles of Fluorescence Spec The fluorescent protein variants of this invention are useful troscopy” (Plenum Press 1983); Herman, “Resonance energy in Systems to detect induction of transcription. For example, 15 transfer microscopy” In “Fluorescence Microscopy of Living a nucleotide sequence encoding a non-oligomerizing mono Cells in Culture' Part B. Meth. Cell Biol. 30:219-243 (ed. meric, dimeric or tandem dimeric fluorescent protein can be Taylor and Wang: Academic Press 1989); Turro, “Modern fused to a promoter or other expression control sequence of Molecular Photochemistry” (Benjamin/Cummings Publ. interest, which can be contained in an expression vector, the Co., Inc. 1978), pp. 296-361, each of which is incorporated construct can be transfected into a cell, and induction of the herein by reference). promoter (or other regulatory element) can be measured by Accordingly, the present invention provides a method for detecting the presence or amount of fluorescence, thereby identifying the presence of a molecule in a sample. Such a allowing a means to observe the responsiveness of a signaling method can be performed, for example, by linking a fluores pathway from receptor to promoter. cent protein variant of the invention to the molecule, and A fluorescent protein variant of the invention also is useful 25 detecting fluorescence due to the fluorescent protein variant in applications involving FRET, which can detect events as a in a sample Suspected of containing the molecule. The mol function of the movement offluorescent donors and acceptors ecule to be detected can be a polypeptide, a polynucleotide, or towards or away from each other. One or both of the donor/ any other molecule, including, for example, an antibody, an acceptor pair can be a fluorescent protein variant. Such a enzyme, or a receptor, and the fluorescent protein variant can donor/acceptor pair provides a wide separation between the 30 be a tandem dimer fluorescent protein. excitation and emission peaks of the donor, and provides The sample to be examined can be any sample, including a good overlap between the donor emission spectrum and the biological sample, an environmental sample, or any other acceptor excitation spectrum. Variant red fluorescent proteins sample for which it is desired to determine whether a particu or red-shifted mutants as disclosed herein are specifically lar molecule is present therein. For example, the sample disclosed as the acceptor in Such a pair. 35 includes a cell or an extract thereof. The cell can be obtained FRET can be used to detect cleavage of a substrate having from a vertebrate, including a mammal Such as a human, or the donor and acceptor coupled to the Substrate on opposite from an invertebrate, and can be a cell from a plant or an sides of the cleavage site. Upon cleavage of the Substrate, the animal. The cell can be obtained from a culture of such cells, donor/acceptor pair physically separate, eliminating FRET. for example, a cell line, or can be isolated from an organism. Such an assay can be performed, for example, by contacting 40 As such, the cell can be contained in a tissue sample, which the Substrate with a sample, and determining a qualitative or can be obtained from an organism by any means commonly quantitative change in FRET (see, for example, U.S. Pat. No. used to obtain a tissue sample, for example, by biopsy of a 5,741,657, which is incorporated herein by reference). A human. Where the method is performed using an intact living fluorescent protein variant donor/acceptor pair also can be cell or a freshly isolated tissue or organ sample, the presence part of a fusion protein coupled by a peptide having a pro 45 of a molecule of interest in living cells can be identified, thus teolytic cleavage site (see, for example, U.S. Pat. No. 5,981, providing a means to determine, for example, the intracellular 200, which is incorporated herein by reference). FRET also compartmentalization of the molecule. The use of the fluo can be used to detect changes in potential across a membrane. rescent protein variants of the invention for Such a purpose For example, a donor and acceptor can be placed on opposite provides a substantial advantage in that the likelihood of sides of a membrane Such that one translates across the mem 50 aberrant identification or localization due to oligomerization brane in response to a Voltage change, thereby producing a the fluorescent protein is greatly minimized. measurable FRET (see, for example, U.S. Pat. No. 5,661,035, A fluorescent protein variant can be linked to the molecule which is incorporated herein by reference). directly or indirectly, using any linkage that is stable under the In other embodiments, a fluorescent protein of the inven conditions to which the protein-molecule complex is to be tion is useful for making fluorescent sensors for protein 55 exposed. Thus, the fluorescent protein and molecule can be kinase and phosphatase activities or indicators for Small ions linked via a chemical reaction between reactive groups and molecules such as Ca", Zn", cyclic 3',5'-adenosine present on the protein and molecule, or the linkage can be monophosphate, and cyclic 3',5'-guanosine monophosphate. mediated by linker moiety, which contains reactive groups Fluorescence in a sample generally is measured using a specific for the fluorescent protein and the molecule. It will be fluorimeter, wherein excitation radiation from an excitation 60 recognized that the appropriate conditions for linking the Source having a first wavelength, passes through excitation fluorescent protein variant and the molecule are selected optics, which cause the excitation radiation to excite the depending, for example, on the chemical nature of the mol sample. In response, a fluorescent protein variant in the ecule and the type of linkage desired. Where the molecule of sample emits radiation having a wavelength that is different interest is a polypeptide, a convenient means for linking a from the excitation wavelength. Collection optics then collect 65 fluorescent protein variant and the molecule is by expressing the emission from the sample. The device can include a tem them as a fusion protein from a recombinant nucleic acid perature controller to maintain the sample at a specific tem molecule, which comprises a polynucleotide encoding, for US 9,075,045 B2 47 48 example, a tandem dimer fluorescent protein operatively The first and second molecules can be cellular proteins that linked to a polynucleotide encoding the polypeptide mol are being investigated to determine whether the proteins spe ecule. cifically interact, or to confirm Such an interaction. Such first A method of identifying an agent or condition that regu and second cellular proteins can be the same, where they are lates the activity of an expression control sequence also is being examined, for example, for an ability to oligomerize, or provided. Such a method can be performed, for example, by they can be different where the proteins are being examined as exposing a recombinant nucleic acid molecule, which specific binding partners involved, for example, in an intrac includes a polynucleotide encoding a fluorescent protein vari ellular pathway. The first and second molecules also can be a ant operatively linked to an expression control sequence, to an polynucleotide and a polypeptide, for example, a polynucle agent or condition Suspected of being able to regulate expres 10 otide known or to be examined for transcription regulatory sion of a polynucleotide from the expression control element activity and a polypeptide known or being tested for sequence, and detecting fluorescence of the fluorescent pro transcription factor activity. For example, the first molecule tein variant due to Such exposure. Such a method is useful, for can comprise a plurality of nucleotide sequences, which can example, for identifying chemical or biological agents, be random or can be variants of a known sequence, that are to including cellular proteins, that can regulate expression from 15 be tested for transcription regulatory element activity, and the the expression control sequence, including cellular factors second molecule can be a transcription factor, Such a method involved in the tissue specific expression from the regulatory being useful for identifying novel transcription regulatory element. As such, the expression control sequence can be a elements having desirable activities. transcription regulatory element Such as a promoter, The present invention also provides a method for determin enhancer, silencer, intron splicing recognition site, polyade ing whether a sample contains an enzyme. Such a method can nylation site, or the like; or a translation regulatory element be performed, for example, by contacting a sample with a Such as a ribosome binding site. tandem fluorescent protein variant of the invention; exciting The fluorescent protein variants of the invention also are the donor, and determining a fluorescence property in the useful in a method of identifying a specific interaction of a sample, wherein the presence of an enzyme in the sample first molecule and a second molecule. Such a method can be 25 results in a change in the degree of fluorescence resonance performed, for example, by contacting the first molecule, energy transfer. Similarly, the present invention relates to a which is linked to a donor first fluorescent protein variant, and method for determining the activity of an enzyme in a cell. the second molecule, which is linked to an acceptor second Such a method can be performed, for example, providing a fluorescent protein variant, under conditions that allow a spe cell that expresses a tandem fluorescent protein variant con cific interaction of the first molecule and second molecule: 30 struct, wherein the peptide linker moiety comprises a cleav exciting the donor, and detecting fluorescence or lumines age recognition amino acid sequence specific for the enzyme cence resonance energy transfer from the donor to the accep coupling the donor and the acceptor; exciting said donor, and tor, thereby identifying a specific interaction of the first mol determining the degree of fluorescence resonance energy ecule and the second molecule. The conditions for Such an transfer in the cell, wherein the presence of enzyme activity in interaction can be any conditions under which is expected or 35 the cell results in a change in the degree of fluorescence Suspected that the molecules can specifically interact. In par resonance energy transfer. ticular, where the molecules to be examined are cellular mol Also provided is a method for determining the pH of a ecules, the conditions generally are physiological conditions. sample. Such a method can be performed, for example, by AS Such, the method can be performed in vitro using condi contacting the sample with a first fluorescent protein variant, tions of buffer, pH, ionic strength, and the like, that mimic 40 which can be a tandem dimer fluorescent protein, wherein the physiological conditions, or the method can be performed in emission intensity of the first fluorescent protein variant a cell or using a cell extract. changes as pH varies between pH 5 and pH 10; exciting the Luminescence resonance energy transfer entails energy indicator, and determining the intensity of light emitted by transfer from a chemiluminescent, bioluminescent, lan the first fluorescent protein variant at a first wavelength, thanide, or transition metal donor to the red fluorescent pro 45 wherein the emission intensity of the first fluorescent protein tein moiety. The longer wavelengths of excitation of red fluo variant indicates the pH of the sample. The first fluorescent rescent proteins permit energy transfer from a greater variety protein variant useful in this method, or in any method of the of donors and over greater distances than possible with green invention, can comprise two DsRed monomers. It will be fluorescent protein variants. Also, the longer wavelengths of recognized that Such fluorescent protein variants similarly are emission is more efficiently detected by solid-state photode 50 useful, either alone or in combination, for the variously dis tectors and is particularly valuable for in Vivo applications closed methods of the invention. where red light penetrates tissue far better than shorter wave The sample used in a method for determining the pH of a lengths. Chemiluminescent donors includebutare not limited sample can be any sample, including, for example, a biologi to luminol derivatives and peroxyoxalate systems. Biolumi cal tissue sample, or a cell or a fraction thereof. In addition, nescent donors include but are not limited to aequorin, obelin, 55 the method can further include contacting the sample with a firefly luciferase, Renilla luciferase, bacterial luciferase, and second fluorescent protein variant, wherein the emission variants thereof. Lanthanide donors include but are not lim intensity of the second fluorescent protein variant changes as ited to terbium chelates containing ultraviolet-absorbing sen pH varies from 5 to 10, and wherein the second fluorescent sitizer chromophores linked to multiple liganding groups to protein variant emits at a second wavelength that is distinct shield the metal ion from solvent water. Transition metal 60 from the first wavelength; exciting the second fluorescent donors include but are not limited to ruthenium and osmium protein variant; determining the intensity of light emitted by chelates of oligopyridine ligands. Chemiluminescent and the second fluorescent protein variant at the second wave bioluminescent donors need no excitation light but are ener length; and comparing the fluorescence at the second wave gized by addition of Substrates, whereas the metal-based sys length to the fluorescence at the first wavelength. The first (or tems need excitation light but offer longer excited state life 65 second) fluorescent protein variant can include a targeting times, facilitating time-gated detection to discriminate sequence, for example, a cell compartmentalization domain against unwanted background fluorescence and scattering. Such a domain that targets the fluorescent protein variant in a US 9,075,045 B2 49 50 cell to the cytosol, the endoplasmic reticulum, the mitochon Kits of the Invention drial matrix, the chloroplast lumen, the medial trans-Golgi The present invention also provides kits to facilitate and/or cisternae, alumen of a lysosome, or a lumen of an endosome. standardize use of compositions provided by the present For example, the cell compartmentalization domain can invention, as well as facilitate the methods of the present include amino acid residues 1 to 81 of human type II mem invention. Materials and reagents to carry out these various brane-anchored protein galactosyltransferase, or amino acid methods can be provided in kits to facilitate execution of the residues 1 to 12 of the presequence of subunit IV of cyto methods. As used herein, the term "kit' is used in reference to chrome c oxidase. a combination of articles that facilitate a process, assay, Detectable Labels Useful for the Invention analysis or manipulation. The detectable label useful according to the invention may 10 Kits can contain chemical reagents (e.g., polypeptides or be selected from the group consisting of radioactive, enzy polynucleotides) as well as other components. In addition, matic, colorimetric, chemiluminescent, fluorescent, electro kits of the present invention can also include, for example but chemical labels and combinations thereof. In an exemplary not limited to, apparatus and reagents for sample collection embodiment of this aspect, the detectable label is a fluores and/or purification, apparatus and reagents for product col cent compound. In an exemplary embodiment, the detectable 15 lection and/or purification, reagents for bacterial cell trans label is selected from the group consisting of fluorescein, formation, reagents for eukaryotic cell transfection, previ rhodamine, bodipy, cyanine, Alexa, Naphthofluorescein, ously transformed or transfected host cells, sample tubes, Oregon Green, coumarin, dansyl, Texas Red, pyrene, Cas holders, trays, racks, dishes, plates, instructions to the kit cade Blue, and Alexa 350 and derivatives thereof. In certain user, Solutions, buffers or other chemical reagents, Suitable embodiments, fluorescent proteins may be used. For samples to be used for standardization, normalization, and/or example, green fluorescent proteins (GFPs) of cnidarians, control samples. Kits of the present invention can also be which act as their energy-transfer acceptors in biolumines packaged for convenient storage and safe shipping, for cence, can be used in the invention. A green fluorescent pro example, in a box having a lid. tein, as used herein, is a protein that fluoresces greenlight, and In some embodiments, for example, kits of the present a blue fluorescent protein is a protein that fluoresces blue 25 invention can provide a fluorescent protein of the invention, a light. GFPs have been isolated from the Pacific Northwest polynucleotide vector (e.g., a plasmid) encoding a fluorescent jellyfish, Aequorea victoria, from the sea pansy, Renilla ren protein of the invention, bacterial cell strains suitable for iformis, and from Phialidium gregarium (Ward et al., 1982, propagating the vector, and reagents for purification of Photochem. Photobiol. 35:803-808: Levine et al., 1982, expressed fusion proteins. Comp. Biochem. Physiol. 72B:77-85). 30 A kit can contain one or more compositions of the inven An exemplary fluorescent protein is green fluorescent pro tion, for example, one or a plurality of fluorescent protein tein (GFP) or a modified GFP. Wild-type GFP has long been variants, which can be a portion of a fusion protein, or one or used in the art. Starting from green fluorescent protein, many a plurality of polynucleotides that encode the polypeptides. modified versions have been derived with altered or enhanced The fluorescent protein variant can be a mutated fluorescent spectral properties as compared with wild-type GFP. See, 35 protein having a reduced propensity to oligomerize, such as a e.g., U.S. Pat. No. 5,625,048; International Patent Publication non-oligomerizing monomer, or can be a tandem dimer fluo WO 97/28261; International Patent Publication WO rescent protein and, where the kit comprises a plurality of 96/23810. Useful are the modified GFPs W1B and TOPAZ, fluorescent protein variants, the plurality can be a plurality of available commercially from Aurora Biosciences Corp., San the mutated fluorescent protein variants, or of the tandem Diego, Calif. W1B contains the following changes from the 40 dimer fluorescent proteins, or a combination thereof. wild-type GFP sequence: F64L, S65T, Y66W7 N1461, A kit of the invention also can contain one or a plurality of M153T, and V163A (see Table 1, page 519, of Tsien, 1998, recombinant nucleic acid molecules, which encode, in part, Ann. Rev. Biochem. 67:509-544). TOPAZ contains the fol fluorescent protein variants, which can be the same or differ lowing changes from the wild-type GFP sequence: S65G, ent, and can further include, for example, an operatively V68L, S72A, and T203Y (see Table 1, page 519, of Tsien, 45 linked second polynucleotide containing or encoding a 1998, Ann. Rev. Biochem. 67:509-544). Wild-type nucle restriction endonuclease recognition site or a recombinase otide and amino acid sequences of GFP are shown in FIG. 1 recognition site, or any polypeptide of interest. In addition, and SEQ ID NO: 1 of International Patent Publication WO the kit can contain instructions for using the components of 97/28261; in FIG. 1 of Tsien, 1998, Ann. Rev. Biochem. the kit, particularly the compositions of the invention that are 67:509544; and in Prasher et al., 1992, Gene 111:229. Of 50 contained in the kit. particular interest in using fluorescent proteins in FRET Such kits can be particularly useful where they provide a based screening assays are variants of the A. Victoria GFP plurality of different fluorescent protein variants because the known as Cyan FP (CFP, Donor (D)) and Yellow FP (YFP. artisan can conveniently select one or more proteins having Acceptor(A)). As an example, the YFP variant can be made as the fluorescent properties desired for a particular application. a fusion protein with, for example, a CKAP4 polypeptide. 55 Similarly, a kit containing a plurality of polynucleotides Vectors for the expression of GFP variants as fusions (Clon encoding different fluorescent protein variants provides tech) as well as fluorophore-labeled compounds (Molecular numerous advantages. For example, the polynucleotides can Probes) are known in the art. When expressing GFPs in mam be engineered to contain convenient restriction endonuclease malian cells, it may be advantageous to construct versions of or recombinase recognition sites, thus facilitating operative the GFPs having altered codons that conform to those 20 60 linkage of the polynucleotide to a regulatory element or to a codons preferred by mammalian cells (Zolotukhin et al., J. polynucleotide encoding a polypeptide of interest or, if Virol. 1996, 70:4646-46754; Yang et al., 1996, Nucl. Acids desired, for operatively linking two or more the polynucle Res. 24:4592-4593). Another way of improving GFP expres otides encoding the fluorescent protein variants to each other. sion in mammalian cells is to provide an optimal ribosome Antibodies and Antibody Compositions binding site by the use of an additional codon immediately 65 Additionally, the present invention includes a purified anti after the starting methionine (Crarneli et al., 1996, Nature body produced in response to immunization with CKAP4, Biotechnology 14:315-319). APF and/or DHHC2, as well as compositions comprising this US 9,075,045 B2 51 52 purified antibody. Antibodies refer to single chain, two-chain, art (see for example Maddox et al., J. Exp. Med. 158, 1211 and multi-chain proteins and glycoproteins belonging to the 1226, 1993). Such immunoassays typically involve the for classes of polyclonal, monoclonal, chimeric, and hetero mation of complexes between the specific protein and its immunoglobulins; it also includes synthetic and genetically antibody and the measurement of complex formation. engineered variants of these immunoglobulins. "Antibody 5 For the purposes of this invention, the term “antibody’. fragment includes Fab, Fab', F(ab')2, and Fv fragments, as unless specified to the contrary, includes fragments which well as any portion of an antibody having specificity toward a bind a polypeptide. Such fragments include Fv, F(ab') and desired target epitope or epitopes. A humanized antibody is F(ab), fragments, as well as single chain antibodies. Further an antibody derived from a non-human antibody, typically more, the antibodies and fragment thereof may be chimeric murine, that retains or Substantially retains the antigen-bind- 10 antibodies, CDR-grafted antibodies or humanised antibodies. ing properties of the parent antibody but which is less immu Antibodies can be produced by any suitable method. nogenic in humans, U.S. Pat. No. 5.530,101, incorporated Means for preparing and characterising antibodies are well herein by reference in its entirety. known in the art, see for example Harlow and Lane (1988) An antibody composition of the present invention is typi Antibodies: A Laboratory Manual, Cold Spring Harbor cally produced by immunizing a laboratory mammal with an 15 Laboratory Press, Cold Spring Harbor, N.Y. For example, an inoculum of the present invention and to thereby induce in the antibody may be produced by raising an antibody in a host mammal antibody molecules having the appropriate polypep animal against the whole polypeptide or a fragment thereof, tide immunospecificity. The polyclonal antibody molecules for example an antigenic epitope thereof, herein after the are then collected from the mammal and isolated to the extent “immunogen'. The fragment may be any of the fragments desired by well known techniques such as, for example, by 20 mentioned herein (typically at least 10 or at least 15 amino immunoaffinity chromatography. The antibody composition acids long) and comprise a polymorphism (Such as any of the so produced can be used in, interalia, the diagnostic methods polymorphisms mentioned herein). and systems of the present invention to detect CKAP4, APF A method for producing a polyclonal antibody comprises and/or DHHC2 in a sample. immunising a Suitable host animal, for example an experi Monoclonal antibody compositions are also contemplated 25 mental animal, with the immunogen and isolating immuno by the present invention. A monoclonal antibody composition globulins from the animal’s serum. The animal may therefore contains, within detectable limits, only one species of anti be inoculated with the immunogen, blood Subsequently body combining site capable of effectively binding CKAP4, removed from the animal and the IgG fraction purified. APF and/or DHHC2. Thus, a monoclonal antibody compo A method for producing a monoclonal antibody comprises sition of the present invention typically displays a single 30 immortalising cells which produce the desired antibody. binding affinity for CKAP4, APF and/or DHHC2 eventhough Hybridoma cells may be produced by fusing spleen cells from it may contain antibodies capable of binding proteins other an inoculated experimental animal with tumour cells (Kohler than CKAP4, APF and/or DHHC2. Suitable antibodies in and Milstein (1975) Nature 256,495-497). monoclonal form, typically whole antibodies, can also be An immortalized cell producing the desired antibody may prepared using hybridoma technology described by Niman et 35 be selected by a conventional procedure. The hybridomas al., Proc. Natl. Sci., U.S.A., 80:4949-4953 (1983), which may be grown in culture or injected intraperitoneally for description is incorporated herein by reference. Briefly, to formation of ascites fluid or into the blood stream of an form the hybridoma from which the monoclonal antibody allogenic host or immunocompromised host. Human anti composition is produced, a myeloma or other self-perpetuat body may be prepared by in vitro immunisation of human ing cell line is fused with lymphocytes obtained from the 40 lymphocytes, followed by transformation of the lymphocytes spleen of a mammal hyperimmunized with a polypeptide of with Epstein-Barr virus. this invention. For the production of both monoclonal and polyclonal The antibody compositions produced by the above method antibodies, the experimental animal is suitably a goat, rabbit, can be used, for example, in diagnostic and therapeutic rat, mouse, guinea pig, chicken, sheep or horse. If desired, the modalities wherein formation of an CKAP4, APF and/or 45 immunogen may be administered as a conjugate in which the DHHC2-containing immunoreaction product is desired. immunogen is coupled, for example via a side chain of one of The invention also provides the use of an antibody specific the amino acid residues, to a suitable carrier. The carrier for an APF polypeptide for the treatment of a CKAP4-related molecule is typically a physiologically acceptable carrier. disease or disorder. The invention also provides the use of an The antibody obtained may be isolated and, if desired, puri antibody specific for a CKAP4 polypeptide for the treatment 50 fied. of an APF-related disease or disorder. ELISA Antibodies may be raised which bind to an APF polypep The present invention also contemplates the direct mea tide, a CKAP4 polypeptide, and/or a DHCC2 polypeptide. Surements of protein expression, such as for example, Antibodies may be raised which bind to both an APF polypep reporter expression, levels in assays. These include the direct tide and a CKAP4 polypeptide. Antibodies may be raised 55 measurement of protein expression using methods such as against APF fusion proteins such as Met-APF. Typically, the ELISA, radioimmunoassay, gel electrophoresis or western antibody “specifically binds” or “is specific for an APF blotting, and immunohistochemistry. polypeptide, a CKAP4 polypeptide, and/or a DHCC2 ELISA techniques are well-known in the art (see, e.g., polypeptide. The antibody may bind to a glycosylation site on Kenney, et al., J. Immunol. 138:4236 (1987), which is incor an APF polypeptide, a CKAP4 polypeptide, and/or a DHCC2 60 porated herein by reference; and Harlow, supra). The ELISA polypeptide. An antibody, or other compound, “specifically protocol involves coating the wells of microtiter (ELISA) binds to a polypeptide or is “specific for a polypeptide when plates with a monoclonal or polyclonal antibody directed to it binds with preferential affinity to the protein for which it is the protein of interest, e.g., an anti-TNF antibody, at a con specific compared to other polypeptides. Such as chemokine centration of between about 0.5-15ug/ml. The antibody solu or G-protein receptor. A variety of protocols for competitive 65 tion is allowed to incubate in the wells for about 12-24 hours binding or immunoradiometric assays to determine the spe at 4.degree.C. in a humid atmosphere. The unbound antibody cific binding capability of an antibody are well known in the is washed away, and the open sites are blocked with an inert US 9,075,045 B2 53 54 protein, such as bovine serum albumin (BSA) in PBS, which or a high-titer polyclonal antiserum. The bound protein-anti is allowed to incubate in the wells for 1-3 hours. The blocking body complex is Subsequently incubated with a second solution is discarded and about 50 ul of the sample kerati labelled antibody specific for the first antibody to form a nocyte supernatant, obtained from the cell pellet of kerati protein-antibody-antibody complex. For example, the second nocytes after centrifugation as described above, is added to labelled antibody may be labelled with a colorimetric label, the ELISA wells. For quantitation the sample solution is e.g., biotin or horseradish peroxidase, or the antibody may be serially diluted in PBS buffer. After an incubation period, radiolabelled, e.g., with 'I, Sor-P. The bound antibody typically one to five hours at 37° C., preferably one to two protein complex is then assayed using the method appropriate hours, more preferably one hour, or about 12 hours at 4°C., for the label as described above. Of course, one of skill in the the plates are again washed and the second antibody directed 10 art will recognize that the protein may be purified and iden to the protein, e.g., a polyclonal rabbit anti-TNF antibody, is tified merely by separation on a suitable gel. Such as sodium applied. After an incubation period, typically one to five dodecylsulfate (SDS) gel if the amount of protein in the hours, or for example one to two hours, or for example one sample is Sufficiently large. hour, the plates are again washed. A biotin-labeled third anti Immunohistochemistry body, directed against the second antibody, is added (e.g., 15 Similar to Western blotting, immunohistochemistry pro goat anti-rabbit) and incubated for about 0.5-1 hour at 37°C. vides information of a qualitative and quantitative nature. The Afterwashing the unbound material, horseradish peroxidase strength of this procedure is that it allows visualization and labeled streptavidin is added and incubated for 0.5-1 hour at localization of the distribution of a specific protein among 37° C. Because the protein is situated between two layers of various cell types or within different regions of a tissue (see, antibodies, this type of assay is often called a sandwich e.g., Griffiths, et al., Br. J. Dermatol. 124:519 (1991), which is ELISA. Subsequent steps using an o-phenylene diamine incorporated herein by reference). In performing this proce (OPD) substrate (e.g. 1 mg/mL OPD/0.3% H2O2/0.1 M cit dure, tissue samples are flash frozen in an embedding com rate buffer) permit color development, with the intensity of pound, e.g., Tissue-Tek OCT (available commercially from color varying according to the amount of TNF specifically Miles, Inc., Elkhart, Ind.), and stored at -70° C. until used. bound. Alternatively, the second enzyme may be biotinylated 25 The tissue is then cut into sections, typically 6 sections, to eliminate the need for a third antibody. Other methods of and placed on microscope slides. The sequence of steps that assay will be apparent to those of skill in the art. The ELISAs follow are quite similar to the procedure involved in the for human and murine TNF are quite sensitive, reliably development of a Western blot. After fixing the tissue in detecting protein concentrations of less than 50 pg/mL. acetone, the slides are incubated with serum, typically goat Radioimmunoassay 30 serum, to block nonspecific binding sites. The samples are Radioimmunoassay may also be used to quantitate the subsequently incubated with a specific antibody directed production of proteins in a manner similar to that just against the protein of interest. After thorough washing, a described for quantitation by ELISA. Generally, a first pro second, labelled antibody, e.g., a biotinylated antibody, is tein specific antibody is attached to a Substrate using standard added and the amount of bound label is quantitated using techniques, such as those described in Harlow or Sambrook, 35 standard techniques. For example, when a biotinylated anti et al., MOLECULAR CLONING: A LABORATORY body is used, avidin conjugated with biotinylated horse radish MANUAL (Cold Spring Harbor 1989), which is incorporated peroxidase is added, followed by incubation with a chro herein by reference. The bound antibody is then exposed to mogenic peroxidase Substrate, to initiate a colorimetric reac the Supernatant taken from keratinocytes cultured withaputa tion. The sample is then counterstained with hematoxylin. tive skin anti-inflammatory Substance. A second antibody, 40 The presence of chromogen, the color of which will depend carrying a radioactive label Such as as I-modified tyrosine is on the particular Substrate being used, is indicative of the added to the bound protein and allowed to incubate as presence of the protein of interest. Care must by taken, how described above. Typically the labelled antibody has a spe ever, to ensure that the staining pattern is specific. A control cific activity of about 5x 106 cpm/ug. The unbound labelled antibody is used for comparison with the anti-protein anti material is washed away and the remaining label quantitated, 45 body. Untreated control tissue or cells are also compared to using standard techniques, e.g., a Scintillation counter. tissues or cells that have been subjected to an inflammatory Gel Electrophoresis and Western Blotting stimulus. As an alternative, the presence of proteins can be assessed Diagnostic Assays by Western blot analysis (see, e.g., Didierjean, et al., J. Invest. It is envisioned that an invitro diagnostic test strip based on Dermatol. 92:809 (1989), which is incorporated herein by 50 the principles of ligand-receptor affinity and immunochro reference). In addition, Western blot analysis can be used to matography will be used to rapidly and accurately identify analyze samples that cannot be accurately tested in the ELI APF in the urine of patients. The name most commonly used SAS and the WEHI assay (below) due to high protein concen to describe this type of “dipstick' assay format is “lateral tration or the presence of various detergents or solubilizing flow”. agents. 55 Most lateral flow test strips such as these are modeled after Generally, this procedure involves electrophoresing the existing immunoassay formats which are typically “sand cell Supernatant samples on reducing or non-reducing SDS wich' assays in which molecules of interest are analyzed by polyacrylamide gels using standard techniques such as those competitive or inhibition assays. Many variations are pos described in Sambrook. The separated protein in the gel is sible, but they all have in common the formation of a complex electrophoretically blotted onto a nitrocellulose membrane, 60 between a particle that is free in the sample stream and a nylon membrane or some other Suitable Support, again using capture reagent that is bound to a membrane at the test line. common techniques. This membrane is first blocked with an While formation of an immunocomplex at the test line is inert protein, then incubated with a solution containing an most commonly used as the result indicator, it is theoretically antibody to the protein of interest, such as one of the antibod possible to achieve a result using any ligand recognition sys ies described above, to bind to the immobilized protein. In an 65 tem where a detector particle becomes bridged to a capture exemplary embodiment the first antibody is a monoclonal reagent on the membrane. The EC50 for APF is very low antibody which has been raised against the denatured protein nanomolar, indicating an affinity for the receptor, CKAP4 US 9,075,045 B2 55 56 that is equivalent to that which occurs in antigen antibody In one embodiment, the Substrate, e.g., the membrane, e.g., interactions. Given this high affinity, CKAP4 or a portion the membrane strip, is, e.g., between about 0.1 and 0.5 inches thereof may be used as the capture reagent for APF in a in width, e.g., between about 0.2 and 0.4, e.g., between about manner analogous to anti-hCG antibodies being the capture 0.25 and 0.3 inches in width, and is, e.g., between about 1 and reagent for hCG in a lateral flow, dipstick-style pregnancy 4 inches in length, e.g., between about 2 and 3 inches in teSt. length. In one embodiment, an agent described herein, e.g., an Various types of detector reagents can be used for the antibody described herein, covers an area of the substrate, visualization of a signal. The most commonly used materials e.g., the membrane, e.g., the membrane strip, that is greater in commercially available tests are latex beads and colloidal than about 0.01 cm2, e.g., greater than about 0.1, 0.5, or 1 gold particles. Other possibilities include enzyme conjugates, 10 cm2. other colloidal metals, dye sacs, fluorescent particles, and In one embodiment, the test device includes a marker indi magnetic particles. cating how far to "dip' the Substrate into a biological sample, Based on current technology, it is envisioned that a lateral e.g., a fluid sample. flow, dipstick-style APF detection system can be used simply In one embodiment, a method described herein employs a as a “Yes/No' indicator or to quantify the abundance of APF 15 dipstick or other test device format to measure the presence, in urine. It is envisioned that a lateral flow, dipstick-style level, expression or activity of a protein described herein, e.g., detection system for APF will be utilized as an over-the an APF polypeptide, a CKAP4 polypeptide, and/or a DHCC2 counter and as a point-of-care (physicians office or hospital) polypeptide. A dipstick or other test device assay can, for options for patients who suspect they may have IC. example, provide a color indication of, or an increased risk The presence, level, expression, or activity of for example, for, interstitial cystitis based upon the levels of a protein an APF polypeptide, a CKAP4 polypeptide, and/or a DHCC2 described herein, such as an APF polypeptide, a CKAP4 polypeptide can be evaluated in a variety of ways well known polypeptide, and/or a DHCC2 polypeptide, in a sample, e.g., in the art, such as immunoassay, e.g., immunoprecipitation, a fluid sample. In one scenario, the dipstick or other test Western blot analysis (immunoblotting), ELISA, fluores device can react to produce one colorifa level of a first protein cence-activated cell sorting (FACS), and bead-based detec 25 is exceeded, a different color if a level of a second protein is tion assays, such as the LumineXR detection technology pro exceeded, a third colorifa level of a third protein is exceeded, vided by the MultiAnalyte Profiling Kit (Luminex and when three levels are exceeded, the three colors will Corporation, Austin, Tex.). Typically, the level of protein combine to yield a separate color that is easily distinguishable and/or activity, e.g., an APF polypeptide, a CKAP4 polypep from the others. A dipstick or other test device-based assay tide, and/or a DHCC2 polypeptide or activity, in a subject 30 optionally includes an internal negative or positive control. sample is compared to the level and/or activity in a control, A dipstick or other test device-based assay could find use in e.g., the level and/or activity in a tissue from a non-disease a clinical setting by quickly and reliably serve as an indication Subject. of or indicating a heightened risk for interstitial cystitis. This Various types of immunoassays are known in the art. One could save valuable time by allowing the physician to initiate example of an immunoassay is a “sandwich type assay, in 35 treatment sooner, thereby minimizing the harmful effects of which a target analyte(s) Such as an APF polypeptide, a the disease. CKAP4 polypeptide, and/or a DHCC2 polypeptide is “sand In another embodiment, the method of the present inven wiched between a labeled antibody and an antibody immo tion may be utilized in combination with a densitometer or bilized onto a solid Support. The assay is read by observing generally a device for measuring light intensity, transmit the presence and amount of antigen-labeled antibody com 40 tance, reflection or refraction, or for measuring the wave plex bound to the immobilized antibody. Another immunoas length of light as a measure of assay result. Such a device can say useful in the methods and kits described herein is a “com be used in a setting Such as a doctors office, a clinic or a petition' type immunoassay, wherein an antibody bound to a hospital. The densitometer or other device can provide rapid Solid Surface is contacted with a sample (e.g., a fluid sample) measurement of the optical density of dipstick or other test containing both an unknown quantity of antigen analyte and 45 device strips that have been contacted with a bodily fluid or with labeled antigen of the same type. The amount of labeled tissue. antigen bound on the solid Surface is then determined to In a preferred embodiment, a change in color, density, or provide an indirect measure of the amount of antigen analyte other parameter can be read by the naked eye. in the sample. Such immunoassays are readily performed in a In a preferred embodiment, the assay can be read without “dipstick” or other test device format (e.g., a flow-through or 50 the addition of a reagent not already on the Substrate. migratory dipstick or other test device design) for convenient Another possible approach to a diagnostic assay includes use, e.g., home use or use by a health care provider. For the use of electrochemical sensor Strips, such as those used for example, numerous types of dipstick immunoassays assays home glucose testing, onto which a sample is placed, and are described in U.S. Pat. No. 5,656,448. which strips include reagents for initiating a reaction when A test device, e.g., a "dipstick, refers to a Substrate, pref 55 wetted by the sample. The sensor strip is inserted into a meter erably a Substrate that is insoluble in aqueous solution, e.g., a that measures, e.g., diffusion-limited current of a reaction fluid sample, onto which an agent described herein, e.g., an species indicative of the analyte of interest, such as an APF agent that detects a protein described herein, e.g., an antibody polypeptide, a CKAP4 polypeptide, and/or a DHCC2 described herein, is immobilized. The agent can be applied as polypeptide. The meter then yields a display indicative of the a layer on the Substrate, or can also penetrate into the Sub 60 concentration of analyte in the sample. strate. A test device can be a substrate, e.g., a membrane, e.g., Tom et al., U.S. Pat. No. 4,366,241, and Zuk, EP-A 0 143 a membrane strip, onto which an agent described herein is 574 describe migration type assays in which a membrane is immobilized. A test device can include a housing for the impregnated with the reagents needed to perform the assay. Substrate, e.g., a membrane, e.g., a membrane Strip, onto An analyte detection Zone is provided in which labeled ana which an agent described herein is immobilized. In one 65 lyte is bound and assay indicia are read. embodiment, the Substrate is a substrate other than glass, and Bernstein, U.S. Pat. No. 4,770,853, May et al., WO is preferably flexible. 88/08534, and Ching et al., EP-A 0299428 describe migra US 9,075,045 B2 57 58 tion assay devices that incorporate within them reagents that ene or polyester. The porous carrier materials may be in fluid have been attached to colored direct labels, thereby permit communication along substantially the whole fluid flow path ting visible detection of the assay results without addition of So as to assist transfer of fluid along the path by capillary further substances. action. The porous carrier materials may function as Solid Valkirs et al., U.S. Pat. No. 4,632,901, disclose a flow Substrates for attachment of reagents or indicator moieties. In through type immunoassay device comprising antibody (spe Some embodiment, the device may have a control Substance cific to a target antigen analyte) bound to a porous membrane that interacts with the sample of the fluid to improve the or filter to which is added a liquid sample. As the liquid flows accuracy of the test. through the membrane, target analyte binds to the antibody. The size and shape of the carrier are not critical and may The addition of sample is followed by addition of labeled 10 vary. The carrier defines a lateral flow path and may be pre antibody. The visual detection of labeled antibody provides sented in the form of one or more elongated Strips or columns. an indication of the presence of target antigen analyte in the In certain embodiments, the porous carrier is one or more sample. elongated Strips of sheet material, or a plurality of sheets Korom et al., EP-A 0299 359, discloses a variation in the making up in combination an elongate strip. One or more flow-through device in which the labeled antibody is incor 15 reaction Zones and detection Zones would then normally be porated into a membrane that acts as a reagent delivery sys spaced apart along the long axis of the strip. However, in some tem. embodiments the porous carrier could, for example be in Baxter et al., EP-A 0 125 118, disclose a sandwich type other sheet forms, such as a disk. In these cases the reaction dipstick immunoassay in which immunochemical compo Zones and detection Zones would normally be arranged con nents such as antibodies are bound to a solid phase. The assay centrically around the center of the sheet, with a sample device is "dipped for incubation into a sample suspected of application Zone in the center of the sheet. In yet other containing unknown antigen analyte. Enzyme-labeled anti embodiments, the carrier is formed of carrier beads, for body is then added, either simultaneously or after an incuba example beads made from any of the materials described tion period. The device next is washed and then inserted into above. The beads may suitably be sized from about 1 a second solution containing a substrate for the enzyme. The 25 micrometer to about 1 mm. The beads may be packed into the enzyme-label, if present, interacts with the Substrate, causing flow path inside the housing, or may be captured or Supported the formation of colored products that either deposit as a on a Suitable porous Substrate Such as a glass fiber pad. precipitate onto the Solid phase or produce a visible color It will be appreciated that the devices in the apparatus change in the Substrate solution. according to the present invention may be adapted to detect Kali et al., EP-A 0282 192, disclose a dipstick device for 30 more than one marker. This can be done by the use of several use in competition type assays. different reagents in a single reaction Zone, or Suitably by the Rounds in U.S. Pat. No. 4.786,589 describes a dipstick provision in a single device of a plurality of lateral flow paths immunoassay device in which the antibodies have been each adapted for detecting a different analyte. In certain labeled with formazan. embodiments, the plurality of lateral flow paths are defined as Detection Device and Kits 35 separate fluid flow paths in the housing, for example the In another aspect, a kit is provided for detecting an APF plurality of lateral flow paths may be radially distributed polypeptide, a CKAP4 polypeptide, and/or a DHCC2 around a sample receiving port. In other embodiments, the polypeptide in a fluid sample, e.g. urine. In some embodi plurality of fluid flow paths are physically separated by the ments, the kit comprises a housing having a chamber consist housing. In yet other embodiments, multiple lateral flow ing of one or more test wells disposed inside the housing and 40 paths (lanes) can be defined in a single lateral flow membrane having an inlet above each well to allow the introduction of by depositing lines of wax or similar hydrophobic material the fluid samples. Each test well may contain the detection between the lanes. reagents, such as, for example, antibodies, for, for example an An absorbent element may also be included in the instant APF polypeptide, a CKAP4 polypeptide, and/or a DHCC2 devices. The absorbent element is a means for drawing the polypeptide. 45 whole sample through the device by capillary attraction. Gen The housing may be at least partially transparent, or may erally, the absorbent element will consist of a hydrophilic have windows provided therein, for observation of an indica absorbent material Such as a woven or nonwoven textile mate tor region that undergoes a color or fluorescence change. The rial, a filter paper or a glass fiber filter. kit may be designed to contain a dipstick, a lateral flow The device may further comprise at least one filtration device, or migration-type device. Preferably, the instant 50 element to remove impurities from the sample before the device is a dipstick as described in the International Patent sample undergoes analysis. The filtration device may for Application Publication WO/2002/033413. In other embodi example comprise a microporous filtration sheet for removal ments, the device is a lateral flow device of the type disclosed, of cells and other particulate debris from the sample. The for example, in U.S. Pat. Nos. 7.297.529; 7,344,893; and filtration device is typically provided upstream of the sample 7,090,803. 55 application Zone of the fluid flow path, for example in the inlet Typically, a lateral flow device may comprise a housing of the housing or in the housing upstream of the inlet. having an inlet for the sample and side walls defining a fluid In certain embodiments, the instant devices may also lateral flow path extending from the inlet. By “lateral flow', it include a control moiety in a control Zone of the device, is meant liquid flow in which the dissolved or dispersed wherein the control moiety can interact with a component of components of the sample are carried, Suitably at Substan 60 the fluid sample to improve the accuracy of the device. Suit tially equal rates, and with relatively unimpaired flow, later ably, the control Zone is adapted to reduce false positive or ally through the carrier. Preferably, the fluid flow path con false negative results. A false negative result could arise for tains one or more porous carrier materials that are various reasons, including (1) the sample is too dilute, or (2) hydrophilic, but preferably do not absorb water. Suitable the sample was too small to start with. In order to address false examples include, but are not limited to, paper, cellulose, 65 negative mechanism, the control Zone may further comprise a nitrocellulose, pressed fibers, including glass fibers, sintered reference assay element for determining the total analyte glass, ceramic or plastic materials, such as nylon, polyethyl content of the sample, that is to say for establishing that the US 9,075,045 B2 59 60 total analyte content or the total protein content of the sample the physician to initiate treatment Sooner, thereby minimizing is higher than a predetermined minimum. It is possible to the harmful effects of the disease. indicate the presence of protein by the use of tetrabromophe In another embodiment, the method of the present inven nol blue, which changes from colorless to blue depending on tion may be utilized in combination with a densitometer in a the concentration of protein present. device for use in a setting Such as a doctors office, a clinic or In addition, the instant device may further comprise a Sam a hospital. The densitometer can provide rapid measurement pling device for collecting a sample of a fluid. To that end, the of the optical density of dipstick or other test device strips that instant device may include a connector for attaching a needle have been contacted with a bodily fluid or tissue. to the device and a plunger for extracting a sample of fluid Other possible approaches include the use of electrochemi from the body of the patient through the needle into the 10 housing of the device. Additional elements of the instant cal sensor Strips, such as those used for home glucose testing, device may include, but are not limited to, a color chart for onto which a sample is placed, and which strips include interpreting the output of the diagnostic device, a wash liquid reagents for initiating a reaction when wetted by the sample. for carrying a sample of fluid through the device, and a pre The sensor Strip is inserted into a meter that measures, e.g., treatment solution containing a reagent for pretreatment of 15 diffusion-limited current of a reaction species indicative of the fluid sample. the analyte of interest, e.g., an APF polypeptide, a CKAP4 In another embodiment, the kit includes a syringe for polypeptide, and/or a DHCC2 polypeptide. The meter then obtaining a sample of fluid wherein, the barrel of the syringe yields a display indicative of the concentration of analyte in is coated with a mix of detection reagents and an appropriate the sample. carrier such that the detection occurs in the barrel once the The kit can also contain a device to obtain a tissue sample, sample of fluid is drawn or within the time necessary for the Such as a cotton Swab or wooden Swab. detection reagents to react with, e.g. an APF polypeptide, a Palmitoylation Assay CKAP4 polypeptide, and/or a DHCC2 polypeptide. Palmitoylation can be measured according to any method The invention also includes kits for detecting the presence known to those skilled in the art. For example, the incorpo of a protein described herein, e.g., an APF polypeptide, a 25 ration of H-palmitate into acid-precipitable protein can be CKAP4 polypeptide, and/or a DHCC2 polypeptide, in a bio used to monitor palmitoylation. Trichloroacetic acid (TCA) logical sample. For example, the kit can include a compound precipitation, followed by Scintillation counting as a means of or agent capable of detecting protein (e.g., an antibody) or measuring covalent protein modification, is well known to mRNA (e.g., a nucleic acid probe) of a protein described those of skill in the art. herein in a biological sample; and a standard. The agent can 30 In another approach, the fluorescence-based assay be coupled to a detectable label, such as a colored, absorbent described by Varner et al. (2002) can be used. The assay or fluorescent label. The kit can also include a positive and a described by Varner et al. uses a myristoylated peptide sub negative control, e.g., a reagent that contains a protein strate, Myr-Gly-Cys, termed Myr-GCK, that mimics the described herein, e.g., an APF polypeptide, a CKAP4 palmitoylation substrate at the N-terminus of the non-recep polypeptide, and/or a DHCC2 polypeptide. 35 tor Src family kinases. The Myr-GCK substrate peptide and The compound or agent can be packaged in a Suitable its synthesis are described by Creaser & Peterson, 2002, J. container. The kit can further comprise instructions for using Am. Chem. Soc. 124: 2444-2445, which is incorporated the kit to evaluate a subject, e.g., for an APF polypeptide, a herein by reference. The structure of the Myr-GCK substrate CKAP4 polypeptide, and/or a DHCC2 polypeptide. peptide, labeled with the NBD fluorophore NBD (7-ni Another embodiment of the present invention is a dipstick 40 trobenz-2-oxa-1,3-diazol-4-yl, available from Molecular or other test device-based kit, suitable for home testing. Such Probes, Inc., Eugene, Oreg.) is as follows: a screening test would provide convenience, privacy and To perform the palmitoylation assay, Myr-GCK eliminate the necessity and cost of visiting a physician for a (10.mu.M) fluorescently labeled with NBD is incubated for 8 screening test, although the dipstick or other test device kit minutes at 37°C. with 50 us of protein from a cellular mem could also be used in a clinical setting. The dipstick or other 45 brane or pellet fraction containing palmitoyl acyltransferase test device kit could be similar to a home pregnancy kit, (see below), in acylation buffer (50 mM citrate, 50 mM phos known to those of skill in the art, and could provide a color phate, 50 mM Tris, 50 mMCAPS at pH 7.2) in a total volume indication of, or an increased risk for, interstitial cystitis of 100 ul. Palmitoyl CoA (20 uM) is then added and the based upon the levels of a protein described herein, e.g., an mixture is incubated at 37° C. for an additional 7.5 minutes. APF polypeptide, a CKAP4 polypeptide, and/or a DHCC2 50 The assay is stopped by extraction in 1.2 ml of CHCl2: polypeptide, in the sample. Such a dipstick or other test methanol: water (2:1:1). The organic fraction is dried under device-based kit could be provided with a small plastic cup N, and then analyzed by HPLC as described below. for collecting and retaining the sample and for conducting the Dried assay extracts are dissolved in 25 ml of DMSO and test. In one scenario, the dipstick or other test device can react resolved on a reverse-phase, wide pore butyl (5 uM, 300 A. to produce one color if a level of a first protein, a different 55 4.6x0.250 mm) HPLC column using an acetonitrile gradient color if a level of a second protein is exceeded, and when both with a flow rate of 1 ml/min. Initially, the mobile phase is levels are exceeded, the two colors will combine to yield a maintained as water/50% CH. Sub.3CN/O.1% TFA for 5 min third color that is easily distinguishable from the others. utes, followed by a 5 minute linear gradient from 50% to In one embodiment, the kit includes at least 1, e.g., at least 100% acetonitrile. The mobile phase is then maintained at 2, 5, 10, 20, 30, or 50, test devices, e.g., dipsticks, e.g., 60 100% acetonitrile for 10 minutes, followed by a linear gradi membranes, e.g., membrane Strips described herein. In one ent from 100% to 50% acetonitrile over 5 minutes. NBD embodiment, the kit contains a container Suitable for collect label is detected by fluorescence at 531 nm upon excitation at ing a fluid sample. 465 nm. The percentage of palmitoylated peptide in the A dipstick or other test device-based assay, similar to that sample is calculated by dividing the peak area corresponding described above, could find use in a clinical setting by quickly 65 to the palmitate-modified peptide by the total peak area cor and reliably provide an indication of oran increased risk for, responding to both palmitoylated and un-palmitoylated pep interstitial cystitis. This could save valuable time by allowing tides. US 9,075,045 B2 61 62 Cell fractions containing palmitoyl acyltransferase for use the individual. The sample may be a blood, urine, saliva, skin, in the palmitoylation assay described above are prepared cheek cell or hair root sample. The sample is preferably a according to the method of Smith et al. (1995, Mol Pharm 47. urine sample. The sample may be processed before the 241-247), essentially as follows: Cultured cells (e.g., HepG2 method is carried out, for example DNA extraction may be or MCF-7) are grown to about 70% confluence in 150 mm 5 carried out, cells may be cultured or a membrane faction may tissue culture dishes and collected by centrifugation. Cells are be prepared from cells. The polynucleotide or protein in the swollen with a buffer containing 10 mM HEPES (pH 7.4), 10 sample may be cleaved either physically or chemically (e.g. mMKC1, 1.5 mMMgCl, and 5uMPMSF for 30 minutes on using a suitable enzyme). In one embodiment the part of ice. The cells are disrupted by homogenization and centri polynucleotide in the sample is copied (or amplified), e.g. by fuged at 5,500 g for 10 minutes at 4°C. to remove nuclei and 10 cloning or using a PCR based method prior to determining the debris. (The nuclei and debris pellet can be assayed as the presence of mutations or polymorphisms. pellet fraction.) The supernatant from this spin is then ultra Diagnostic Use centrifuged at 100,000 g for 1 hour at 4°C. The pellet from In another embodiment of the present invention, measure this centrifugation is resuspended in 100 ul of lysis buffer and ment of CKAP4, APF and/or DHHC2, or proteins which are collected as the membrane fraction. The Supernatant is col 15 immunologically related to CKAP4, APF and/or DHHC2, lected as the cytosolic fraction. Protein concentrations for can be used to detect and/or stage a disease or disorder in a each fraction are determined (e.g., using a fluorescamine Subject. The measured amount may be compared to a baseline assay, Bohlen et al., 1973, Arch Biochem Biophys 155,213 level. This baseline level can be the amount which is estab 220). lished to be normally present in the body fluid of subjects with Methods of Diagnosis various degrees of the disease or disorder. An amount present The invention further provides methods of diagnosing a in the body fluid of the subject which is similar to a standard CKAP4-related disease or disorder oran APF-related disease amount, established to be normally present in the body fluid or disorder in an individual, such as IC/PBS. A CKAP4- or of the Subject during a specific stage of the disease or disorder, APF-related disease or disorder is typically a disease or dis is indicative of the stage of the disease in the subject. The order is for example, IC/PBS. 25 baseline level could also be the level present in the subject In a diagnostic embodiment the invention, the method of prior to the onset of disease or the amount present during diagnosis comprises contacting a sample isolated from an remission of disease, or from individuals not afflicted with the individual which comprises a CKAP4 peptide with an APF disease or condition. polypeptide under conditions which permit the binding of the The present invention also provides for the detection or APF polypeptide to the CKAP4 polypeptide. The activity of 30 diagnosis of disease or the monitoring of treatment by mea the CKAP4 polypeptide is then measured. The activity of this suring the amounts of CKAP4, APF and/or DHHC2 transcript CKAP4 polypeptide is then compared with a standard and a or peptide in a sample before and after treatment, and com difference in the activity relative to the standard indicative of paring the two measurements. The change in the levels of the the presence of a CKAP4-related disease or disorder in the markers relative to one another can be an improved prognos individual. This standard refers to the equivalent measure 35 tic indicator. A comparison of the amounts of a total marker ment in an individual not affected by the CKAP4-relevant with the amount of intra-cytoplasmic marker or membrane disease or disorder. bound marker is also envisioned. The methods of the diagnostic embodiment also comprise The present invention provides a method for monitoring contacting a 5 sample isolated from an individual which the effect of a therapeutic treatment on a subject who has comprises a APF polypeptide with a CKAP4 polypeptide 40 undergone the therapeutic treatment. This method comprises under conditions which permit the binding of the APF measuring at Suitable time intervals the amount of a soluble polypeptide to the CKAP4 polypeptide. The activity of the molecule or soluble fragment thereof, or the amount of CKAP4 polypeptide is then measured. The activity of this CKAP4, APF and/or DHHC2 or fragment thereof. Any CKAP4 polypeptide is then compared with a standard and a change or absence of change in the amount of the soluble difference in the activity relative to the standard indicative of 45 molecule or in the amount of the CKAP4, APF and/or the presence of a APF-related disease or disorder in the indi DHHC2 can be identified and correlated with the effect of the vidual. This standard refers to the equivalent measurement in treatment on the subject. In a specific embodiment of the an individual not affected by the APF-relevant disease or invention, Soluble molecules immunologically related to disorder. CKAP4, APF and/or DHHC2 can be measured in the serum The conditions which permit the binding of a APF 50 of patients by a sandwich enzyme immunoassay (for an polypeptide to a CKAP4 polypeptide are, for example, the example) in order to predict disease prognosis, for example, temperature, salt concentration, pH and protein concentration in viral infections, inflammation, autoimmune diseases, and under which a APF polypeptide binds to a CKAP4 polypep tumors, or to monitor the effectiveness of treatments such as tide. Exact binding conditions will vary depending on nature anti-viral administration. of the assay, for example, when the assay uses viable cells or 55 FRET-Based Screening Methods only membrane fraction of cells. However, because CKAP4 The physical proximity of the two fluorescent proteins is a cell surface receptor and APF polypeptides are secreted results in increased fluorescence resonance energy transfer, polypeptides that interact with the extracellular domain of which can be detected using fluorescent methods, including CKAP4, conditions will generally include physiological salt FRET microscopy, ratio imaging, or rationnetric fluorimetry. concentration (approx 90 mM) pH about (7.0 to 8.0). Tem 60 An instrument such as FLIPRTM can be set to alternate peratures for binding may vary from 4°C. through to 37°C., between reading signals at two different wavelengths with a but is preferably 4°C. The concentration of the APF polypep cycling time of about one second, and is therefore extremely tide will also vary, but will for example be from about 0.1 uM useful in measuring samples in high-throughput. to about 10 uM. Fluorescence resonance energy transfer (FRET) is a non The methods of the diagnostic embodiments may be, for 65 radiative process whereby energy from a fluorescent donor example, carried out in vitro on a sample from the individual. molecule is transferred to an acceptor molecule without the The sample typically comprises a body fluid and/or cells of involvement of a photon. Excitation of the donor molecule US 9,075,045 B2 63 64 enhances the fluorescence emission of the longer-wavelength No. P42212). Similarly, the fluorescent protein may be acceptor molecule (i.e., sensitized acceptor emission). The related to Renilla or Phialidium wild-type fluorescent pro quantum yield of the donor fluorescence emission is con teins using the same standards. Aequorea-related fluorescent comitantly diminished. FRET has become a valuable tool for proteins include, for example, wild-type (native) Aequorea microscopy, because the efficiency of energy transfer has a victoria GFP, whose nucleotide and deduced amino acid strong inverse dependence on the distance between the donor sequences are presented in GenBank Accession Nos. L29345, and acceptor molecules. Thus, the appearance of FRET is a M62654, M62653 and other Aequorea-related engineered highly specific indicator of the proximity of the two mol versions of Green Fluorescent Protein, of which some are ecules. This has led to the use of FRET efficiency as a “spec listed above. Several of these, i.e., P4, P4-3, W7 and W2 troscopic ruler to measure molecular distances. 10 The recent availability of green fluorescent protein (GFP) fluoresce at a distinctly shorter wavelength than wild type. mutants with shifted excitation and emission spectra has Recombinant nucleic acid molecules encoding single- or made it feasible to measure protein-protein interactions by tandem fluorescent protein/polypeptide comprising engi using GFP tags as intracellular markers. GFP-tagged protein neered binding domain, sequences or polypeptides or their chimeras are expressed intracellularly and do not require any 15 binding partners useful in the invention may be expressed for chemical treatment to become fluorescent. FRET can also in vivo assays of the activity of a modifying enzyme on the occur between fusions of blue-emitting and green-emitting encoded products. GFP variants. Similar assays using different ligands, different detection As described above, a donor fluorescent protein label is techniques, etc. are readily designed by one of skill in the art capable of absorbing a photon and transferring energy to using the information provided in the art generally. another fluorescent label. The acceptor fluorescent protein Isosteric Binding Molecule label is capable of absorbing energy and emitting a photon. If There are a variety of techniques well known in the art for needed, the linker connects the binding domain, sequence or assessing compound modulation of APF binding to CKAP4. polypeptide either directly, or indirectly through an interme A wide variety of detectable labels can be used, and the assays diary linkage, with one or both of the donor and acceptor 25 can be conducted in heterogeneous or homogeneous formats. fluorescent protein labels or the fluorescent label and, option The assay can be conducted in a homogeneous format using ally, the quencher if a non-FRET assay is being performed. fluorescence techniques, for example, where the fluorescence Regardless of the relative order of the binding domain, of a fluorescent label attached to the compound is altered by sequence or polypeptide or its binding partner and the donor virtue of its binding to a peptide the size of the CKAP4, APF and acceptor fluorescent protein labels on a polypeptide mol 30 and/or DHHC2, or larger. The CKAP4 itself can be supplied ecule, it is essential that sufficient distance be placed between in the context of the nonstructural protein or fragment thereof the donor and acceptor or the fluorescent label and corre or can be supplied perse or as a fusion protein that contains a sponding quencher by the linker and/or the binding domain, label, such as green fluorescent protein. sequence, nucleic acid or polypeptide and corresponding In an exemplary extracellular format, the effect of indi binding partner to ensure that FRET does not occur unless the 35 vidual compounds or mixtures of compounds on the binding binding domain, sequence or polypeptide and its binding of the APF or a protein containing it with a CKAP4 containing partner bind. It is desirable, as described in greater detail in membrane preparation can be evaluated. Techniques similar WO97/28261, to select a donor fluorescent protein label with to those described above may be used—e.g., the CKAP4 an emission spectrum that overlaps with the excitation spec and/or the membrane preparation may be labeled and the APF trum of an acceptor fluorescent protein label. In some 40 or protein containing it. The binding of the label APF to embodiments of the invention the overlap in emission and CKAP4 in the presence and absence of compound or mix excitation spectra will facilitate FRET. A fluorescent protein tures of compounds can be determined. These assays can be of use in the invention includes, in addition to those with conducted in homogeneous format by using fluorescence intrinsic fluorescent properties, proteins that fluoresce due to labeling, for example, of the CKAP4, APF and/or DHHC2. intramolecular rearrangements or the addition of cofactors 45 A wide variety of such protocols is known in the art, and the that promote fluorescence. essential feature is determination of the effect of the com For example, green fluorescent proteins (GFPs) of cnidar pound or a mixture of compounds on the binding of the APF ians, which act as their energy-transfer acceptors in biolumi or a protein containing it and the CKAP4 containing mem nescence, can be used in the invention. A green fluorescent brane preparation. Perhaps a particularly convenient embodi protein, as used herein, is a protein that fluoresces greenlight, 50 ment of this method would involve a fusion between the and a blue fluorescent protein is a protein that fluoresces blue CKAP4 and a fluorescent protein which could be produced light. GFPs have been isolated from the Pacific Northwest very conveniently using recombinant techniques; assessment jellyfish, Aequorea victoria, from the sea pansy, Renilla ren of the binding of APF to this fusion in the membrane prepa iformis, and from Phialidium gregarium (Ward et al., 1982, ration either in a heterogeneous or homogeneous format can Photochem. Photobiol. 35:803-808: Levine et al., 1982, 55 then be performed. Comp. Biochem. Physiol. 72B:77-85). A variety of Aequo An assay for binding of the APF or APF containing peptide rea-related GFPs having useful excitation and emission spec to a CKAP4 containing microsomal membrane can be per tra have been engineered by modifying the amino acid formed by treating a microsomal or cytoplasmic membrane sequence of a naturally-occurring GFP from Aequorea Victo preparation in vitro with a peptide containing the APF and ria (Prasher et al., 1992, Gene 111:229-233; Heim et al., 60 distributing the contents of the reaction mixture in a sedimen 1994, Proc. Natl. Acad. Sci. U.S.A. 91: 12501-12504; tation gradient. The bound APF can be detected in the appro PCTUS95/14692). As used herein, a fluorescent protein is an priate gradient fraction using polyacrylamide gel electro Aequorea-related fluorescent protein if any contiguous phoresis. The bound APF and/or CKAP4 may be labeled for sequence of 150 amino acids of the fluorescent protein has at example, with a radioisotope or a coupled fluorescent label least 85% sequence identity with an amino acid sequence, 65 Such as green fluorescent protein used in a fusion. This assay either contiguous or non-contiguous, from the wild-type can be used to screen for compounds or protocols that disrupt Aequorea green fluorescent protein (SwissProt Accession binding to the microsomal membrane by conducting the US 9,075,045 B2 65 66 assay in the presence and absence of the protocols or com addition to causing the protein to fail to associate with pounds and comparing the results. CKAP4 containing cytoplasmic membranes, directs this pro In addition to the above-described methods that can be tein to the nucleus. Therefore, the assay methods can include conducted extracellularly, the effect of various protocols and features in the substance containing the CKAP4 that act as compounds or mixtures of compounds on the behavior in 5 nuclear localization signals (NLS), many of which are well terms of binding to CKAP4 containing cytoplasmic mem known in the art. Under these conditions the label can con branes of the APF or APF containing peptide can be deter stitute a functionality which exerts its effect in the nucleus, mined in a variety of intracellular assays. Any eukaryotic cells and the disruption of binding of the APF to CKAP4 contain may be used, but typically and most conveniently, mamma ing cytoplasmic membrane can be detected by a reporter lian cells or yeast cells are used in these assays. 10 function associated with the APF to CKAP4 binding which Intracellular assays can be performed by generating exerts its effects in the nucleus. Such effects would be, for desired peptide constructs intracellularly from recombinant example, enhancement or repression of the expression of a expression systems. Alternatively, the assays can be con detectable protein; this would involve including transactiva ducted by first preparing the labeled CKAP4, APF and/or tors, transcription factors, or repressors, and the NLS. Of DHHC2 or protein containing said CKAP4, APF and/or 15 course, the nuclear localization signal—derived constructs DHHC2 and introducing the derivatized CKAP4, APF and/or can also be detected in the nucleus directly, using a direct DHHC2 into the cells using cell-penetrating peptides. The detectable label of the sort described above e.g., GFP, a compounds to be tested may be introduced in the same man partner in an antibody/antigen interaction, or an enzymatic ner. Such cell-penetrating peptides are described, for activity, or a radiolabel. example, in a review article by Lindgrin, M., et al., in TiPS Alternative proteins whose expression can be detected are (2000) 21:99-103, the contents of which are incorporated those, such as GFP, that are detectable per se or may be herein by reference to describe an exemplary list of such detectable by virtue of their effects on cellular growth, such as cell-penetrating peptides. These peptides can be coupled to his3, leu2, B-galactosidase, B-glucuronidase, SV40T antigen, any substance to facilitate the entry of said substance into a chloramphenicol acetyl transferase (CAT), hygromycin B eukaryotic cell. 25 phosphotransferase, SEAP or cell Surface antigens such as In the most direct forms of Such assays, the change in CD4. In addition to transactivators, transcription factors intracellular distribution of the labeled APF and/or CKAP4 which enhance expression include derivatives of lexa, cI, or either Supplied perse or in the context of a larger protein can galA DNA binding domains fused to activation domains of be determined. A wide variety of labels can be used; perhaps B42, VP16 and the like. the most convenient is a fusion with a fluorescent protein, or 30 By conducting the foregoing assays, compounds and/or simple coupling of the CKAP4 or the protein containing it to protocols are identified which will be effective in treating IC. a detectable fluorescent label. The location of the labeled By “treating is meant both therapeutic and prophylactic CKAP4 or protein containing it can then be observed by a treatment, and refers to a desirable effect on viral load, or variety of methods including direct observation and histologi symptomology, or any desirable outcome which mitigates the cal techniques. Thus, in one example, the effect of a candidate 35 negative consequences of the typical progress of IC infection. compound or protocol directly on the ability of the APF to The term “treat is not to be construed to imply absolute cure bind to CKAP4 containing cellular membranes can be or absolute prevention. Any helpful amelioration or repres assessed by coupling the CKAP4 to a reporter which is detect sion of the infection is sufficient to meet this definition. able; most convenient are labels which are fusion proteins Compounds thus identified can be administered in conven formed with the CKAP4, such as a fluorescent protein. The 40 tional ways using standard pharmaceutical formulations such intracellular locations of the reporter in the presence and as those set forth in Remington's Pharmaceutical Sciences, absence of the candidate protocol compound can then be latest edition, Mack Publishing Co., Easton, Pa., incorporated compared. Compounds or protocols that are able to disrupt herein by reference. (See also, References 23-33.) Various the binding of the APF with the CKAP4 in the membrane can enteral or parenteral routes of administration may be used, readily be identified when their presence results in less label 45 including administering by injection, such as intravenous, associated with cell membranes. intramuscular, Subcutaneous and the like, or by oral admin If histological techniques are used, the label can be less istration or by Suppository. In addition, Sustained release direct; for example, the CKAP4 might be fused to a protein compositions can also be used. that can be detected with a labeled antibody or may be fused The terms “an effective amount,” “an anti-IC effective to an enzyme that can be detected in the presence of a Sub 50 amount,” or a “pharmaceutically effective amount of a pep strate. Once the cells are fixed histologically, the Supplemen tide of the present invention as applied to Such molecules tary reagents can be added for detection. refers to an amount of a peptide, or combination of two or Test compounds that appear to interrupt the ability of the more peptides as disclosed herein, effective in reducing or APF to bind CKAP4 in the cellular membranes, or which are ameliorating conditions, symptoms, or disorders associated shown to disrupt the normal binding pattern of the APF to 55 with IC or associated pathogenesis in patients. CKAP4 can be confirmed as anti-IC agents. Effective amounts of compounds for the treatment or pre When intracellular formats are used, in addition to testing vention of IC, delivery vehicles containing compounds or compounds and mixtures of compounds, as noted above, it is constructs encoding the same, agonists, and treatment proto possible to test the effect of protocols which involve various cols, can be determined by conventional means. For example, regimes of treatment, including, in addition to providing com 60 the medical practitioner can commence treatment with a low pounds, various antisense techniques, and various forms of dose of one or more peptides in a subject or patient in need environmental stress Such as pH changes, temperature thereof, and then increase the dosage, or systematically vary changes, mechanical disturbances and the like. the dosage regimen, monitor the effects thereof on the patient In addition to the foregoing, somewhat more Sophisticated or Subject, and adjust the dosage or treatment regimen to methods to assess the impact of compounds and protocols on 65 maximize the desired therapeutic effect. Further discussion of the intracellular location of substances containing the APF optimization of dosage and treatment regimens can be found and/or CKAP4 proteins can be employed. For example, in in Benet et al., in Goodman & Gilman's The Pharmacological US 9,075,045 B2 67 68 Basis of Therapeutics, Ninth Edition, Hardman et al., Eds. CKAP4, APF and/or DHHC2 encompassed by the present McGraw-Hill, New York, (1996), Chapter 1, pp. 3-27, and L. invention refers to peptide sequences like those disclosed A. Bauer, in Pharmacotherapy, A Pathophysiologic herein, but which contain additional amino acids (or analogs Approach, Fourth Edition, DiPiro et al., Eds. Appleton & or derivatives thereof as discussed above). Such additional Lange, Stamford, Conn., (1999), Chapter 3, pp. 21-43, and amino acids, etc., however, do not materially affect the basic the references cited therein, to which the reader is referred. and novel characteristic(s) of these peptides in modulating, The dosage levels and mode of administration will be attenuating, or inhibiting IC infection, replication, and/or dependent on the nature of the compound identified and the pathogenesis, including the specific quantitative effects of particular situation of the subject. Optimization of routes of these peptides, compared to those of the corresponding pep administration, dosage levels, and adjustment of protocols, 10 tides disclosed herein. including monitoring systems to assess effectiveness of the siNA treatment are routine matters well within ordinary skill. Pro This invention comprises compounds, compositions, and tocols which are identified using the intracellular assays set methods useful for modulating CKAP4 and/or APF gene or forth above must, of course, be modified in the context of promoter expression or activity using short interfering treatment of subjects, and this, too, falls within the skill of the 15 practitioner. nucleic acid (siNA) molecules. This invention also comprises Typically, the compounds that are identified by the meth compounds, compositions, and methods useful for modulat ods of the invention will be “small molecules’ i.e., syn ing the expression and activity of other genes involved in thetic organic structures typical of pharmaceuticals. pathways of, for example, CKAP4, APF, and/or DHHC2 Examples of such “small molecules' are found, for example, gene, or promoter expression and/or activity by RNA inter in the Physicians’ Desk Reference (with respect to approved ference (RNAi) using Small nucleic acid molecules. In par drugs), the Merck Index, and the U.S. Pharmacopoeia. How ticular, the instant invention features Small nucleic acid mol ever, such compounds may also include peptides, peptidomi ecules, such as short interfering nucleic acid (siNA), short metics, nucleic acids, peptide nucleic acids, carbohydrates, interfering RNA (siRNA), double-stranded RNA (dsRNA), lipids, and the like. 25 micro-RNA (miRNA), and short hairpin RNA (shRNA) mol In addition to compounds identified by means of the fore ecules and methods used to modulate the expression or activ going assays, protocols and Substances useful in treating IC ity of for example, CKAP4, APF, and/or DHHC2 gene, or can beformulated and utilized a priori. For example, the APF, promoter, or the activity or expression of other components of the CKAP4 protein, or peptides thereof protein, or functional the pathway. fragment thereof can readily be used in treatment by virtue of 30 A siNA of the invention can be unmodified or chemically its ability to bind CKAP4 or to compete with the APF binding modified. A siNA of the instant invention can be chemically to CKAP4. synthesized, expressed from a vector or enzymatically syn Thus, formulations of the CKAP4, APF and/or DHHC2 thesized. The instant invention also features various chemi protein, or peptides thereof protein, or functional fragment cally-modified synthetic short interfering nucleic acid (siNA) thereof can be used directly to treat IC infection. Typically, 35 molecules capable of modulating CKAP4 and/or APF gene or these formulations will contain peptides of less than 60 amino promoter expression or activity in cells by RNA interference acids, in another embodiment less than 50 amino acids, in (RNAi). The use of chemically-modified siNA improves vari another embodiment less than 30 amino acids, and, in yet ous properties of native siNA molecules through increased another embodiment less than 25 amino acids in another resistance to nuclease degradation in vivo and/or through embodiment, not be less than 4 amino acids. The peptides 40 improved cellular uptake. Further, contrary to earlier pub may be as short as 10-15 amino acids, 6-9 amino acids or 4-6 lished studies, siNA having multiple chemical modifications amino acids, so long as functionality in terms of competitive retains its RNAi activity. The siNA molecules of the instant binding, or inhibition of binding, is retained. Methods to invention provide useful reagents and methods for a variety of synthesize such peptides are, of course, well known, both therapeutic, diagnostic, target validation, genomic discovery, direct and recombinant methods may be used. Thus, a peptide 45 genetic engineering, and pharmacogenomic applications. useful in the method of the invention will typically contain In one embodiment, the invention features a double from about 4 to 60 amino acids. stranded short interfering nucleic acid (siNA) molecule that The peptides useful in the methods of the invention can also modulates, for example, CKAP4, APF, and/or DHHC2 gene, be generated in the subject to be treated by virtue of admin or promoter expression and/or activity, wherein said siNA istering expression systems for those peptides consisting 50 molecule comprises about 19 to about 21 base pairs. entirely of gene-encoded amino acids. These expression sys In one embodiment, the invention features a siNA mol tems may be introduced as naked DNA, as expression vectors ecule that modulates, for example, CKAP4, APF, and/or Suitable for transfection of mammalian cells, or for example DHHC2 gene, or promoter expression and/or activity, for using adenoviral or retroviral or other suitable viral vectors. example, wherein the CKAP4, APF, and/or DHHC2 gene or As described above, however, these competitor peptides 55 promoter comprises CKAP4, APF, and/or DHHC2 encoding need not be the native sequences perse and need not even be sequence. In one embodiment, the invention features a siNA peptides per se, but may contain isosteric linkages or other molecule that modulates expression of a CKAP4, APF, and/or polymeric features that result in similar charge? shape features DHHC2 gene or CKAP4, APF, and/or DHHC2 promoter, for as compared to the native helices. example, wherein the CKAP4, APF, and/or DHHC2 gene or Peptides, or compounds with similar charge/shape features 60 promoter comprises CKAP4, APF, and/or DHHC2 non-cod and having the activity of the peptides described herein, can ing sequence or regulatory elements involved in CKAP4, be identified by phage display using wild-type or mutant APF APF, and/or DHHC2 gene or promoter expression. and wild-type or mutant CKAP4 peptides as selectors. In one embodiment, the invention features a siNA mol The compositions or agents of the invention may comprise, ecule having RNAi activity against, for example, CKAP4, consist essentially of, or consist of the peptide sequences 65 APF, and/or DHHC2, wherein the siNA molecule comprises disclosed herein. The phrase “consists essentially of or con a sequence complementary to any RNA having CKAP4, APF, sisting essentially of or the like, when applied to anti and/or DHHC2 encoding sequence. US 9,075,045 B2 69 70 In another embodiment, the invention features a siNA mol otides, “universal base' nucleotides, “acyclic” nucleotides, ecule having RNAi activity against CKAP4, APF, and/or 5-C-methyl nucleotides, and terminal glyceryl and/or DHHC2 RNA, wherein the siNA molecule comprises a inverted deoxy a basic residue incorporation. These chemical sequence complementary to an RNA having other CKAP4, modifications, when used in various siNA constructs, are APF, and/or DHHC2 encoding sequence, for example other shown to preserve RNAi activity in cells while at the same mutant CKAP4, APF, and/or DHHC2 gene or promoters. time, dramatically increasing the serum stability of these In one embodiment of the invention a siNA molecule com compounds. Furthermore, contrary to the data published by prises an antisense Strand comprising a nucleotide sequence Parrish et al., Supra, applicant demonstrates that multiple that is complementary to a nucleotide sequence or a portion (greater than one) phosphorothioate Substitutions are well thereof encoding, for example, an CKAP4, APF, and/or 10 tolerated and confer Substantial increases in serum stability DHHC2 protein. The siNA further comprises a sense strand, for modified siNA constructs. wherein said sense strand comprises a nucleotide sequence of In one embodiment, a siNA molecule of the invention an CKAP4, APF, and/or DHHC2 gene or promoter or a por comprises modified nucleotides while maintaining the ability tion thereof. to mediate RNAi. The modified nucleotides can be used to In another embodiment, a siNA molecule comprises an 15 improve in vitro or in vivo characteristics such as stability, antisense region comprising a nucleotide sequence that is activity, and/or bioavailability. For example, a siNA molecule complementary to a nucleotide sequence encoding an of the invention can comprise modified nucleotides as a per CKAP4, APF, and/or DHHC2 protein or a portion thereof. centage of the total number of nucleotides present in the siNA The siNA molecule further comprises a sense region, wherein molecule. As such, a siNA molecule of the invention can said sense region comprises a nucleotide sequence of an generally comprise about 5% to about 100% modified nucle CKAP4, APF, and/or DHHC2 gene or promoter or a portion otides (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, thereof. 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, In one embodiment, a siNA molecule of the invention has 95% or 100% modified nucleotides). The actual percentage of RNAi activity that modulates expression of RNA encoded by, modified nucleotides present in a given siNA molecule will for example, an CKAP4, APF, and/or DHHC2 gene. Because 25 depend on the total number of nucleotides present in the CKAP4, APF, and/or DHHC2 gene can share some degree of siNA. If the siNA molecule is single stranded, the percent with each other, siNA molecules can be modification can be based upon the total number of nucle designed to target a class of CKAP4, APF, and/or DHHC2 otides present in the single stranded siNA molecules. Like gene or alternately specific CKAP4, APF, and/or DHHC2 wise, if the siNA molecule is double stranded, the percent gene (e.g., polymorphic variants) by selecting sequences that 30 modification can be based upon the total number of nucle are either shared amongst different CKAP4, APF, and/or otides present in the sense Strand, antisense strand, or both the DHHC2 targets or alternatively that are unique for a specific sense and antisense strands. CKAP4, APF, and/or DHHC2 target. Therefore, in one As used herein, the term “hybridizes under low stringency, embodiment, the siNA molecule can be designed to target medium stringency, high Stringency, or very high Stringency conserved regions of, for example, CKAP4, APF, and/or 35 conditions’ describes conditions for hybridization and wash DHHC2 RNA sequences having homology among several ing. Guidance for performing hybridization reactions can be CKAP4, APF, and/or DHHC2 gene or promoter variants so as found in Current Protocols in Molecular Biology, John Wiley to target a class of CKAP4, APF, and/or DHHC2 gene with & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by one siNA molecule. Accordingly, in one embodiment, the reference. Aqueous and nonaqueous methods are described in siNA molecule of the invention modulates the expression of 40 that reference and either can be used. Specific hybridization for example, one or both CKAP4, APF, and/or DHHC2 alleles conditions referred to herein areas follows: 1) low stringency in a subject. In another embodiment, the siNA molecule can hybridization conditions in 6x sodium chloride/sodium cit be designed to target a sequence that is unique to a specific rate (SSC) at about 45° C., followed by two washes in 0.2x CKAP4, APF, and/or DHHC2 RNA sequence (e.g., a single SSC, 0.1% SDS at least at 50° C. (the temperature of the CKAP4, APF, and/or DHHC2 allele or CKAP4, APF, and/or 45 washes can be increased to 55° C. for low stringency condi DHHC2 SNP) due to the high degree of specificity that the tions); 2) medium Stringency hybridization conditions in siNA molecule requires to mediate RNAi activity. 6xSSC at about 45° C., followed by one or more washes in In one embodiment, nucleic acid molecules of the inven 0.2xSSC, 0.1% SDS at 60° C.; 3) high stringency hybridiza tion that act as mediators of the RNA interference gene silenc tion conditions in 6xSSC at about 45°C., followed by one or ing response are double-stranded nucleic acid molecules. In 50 more washes in 0.2xSSC, 0.1% SDS at 65° C.; and preferably another embodiment, the siNA molecules of the invention 4) very high stringency hybridization conditions are 0.5M consist of duplexes containing about 19 base pairs between sodium phosphate, 7% SDS at 65° C., followed by one or oligonucleotides comprising about 19 to about 25 (e.g., about more washes at 0.2xSSC, 1% SDS at 65° C. 18, 19, 20, 21, 22, 23, 24, 25 or 26) nucleotides. In yet another The invention will be illustrated in more detail with refer embodiment, siNA molecules of the invention comprise 55 ence to the following Examples, but it should be understood duplexes with overhanging ends of about 1 to about 3 (e.g., that the present invention is not deemed to be limited thereto. about 1, 2, or 3) nucleotides, for example, about 21-nucle otide duplexes with about 19 base pairs and 3'-terminal mono EXAMPLES nucleotide, dinucleotide, or trinucleotide overhangs. In one embodiment, the invention features one or more 60 Example 1 chemically-modified siNA constructs having specificity, for example, for CKAP4, APF, and/or DHHC2 expressing The fluorescent reporter CKAP4-YFP was composed of nucleic acid molecules, such as RNA encoding a CKAP4, the following DNA elements: CKAP4 was cloned by reverse APF, and/or DHHC2 protein. Non-limiting examples of such transcription from a HeLa cellcNA library (see FIG.1). The chemical modifications include without limitation phospho 65 5' primer incorporated a kpnI site and the 3' primer an XhoI rothioate internucleotide linkages. 2'-deoxyribonucleotides, site in order to facilitate cloning. YFP was amplified by PCR 2'-O-methyl ribonucleotides. 2'-deoxy-2'-fluoro ribonucle from a plasmid containing the coding sequence for YFP. To US 9,075,045 B2 71 72 aid in cloning the 5' primer forYFP included an XhoI site and CKAP4 expression. Particular care will be taken to choose the 3' primer an EcoRI site. The XhoI endonuclease sites, one clones that do not expressively high or low quantities of the at the 3' end of CKAP4 and the other at the 5' end of YFP reporter. Once clones have been selected for appropriate resulted in an in-frame fusion of CKAP4 to YFP. Both of these expression by visual inspection, they will be characterized cDNA fragments were ligated into pcDNA3 that was linear 5 further by measuring their positive response to Saturating ized with KpnI and EcoRI. Translation of the mRNA gener concentration of synthetic APF (defined here as 20 nM or ated from this expression construct began at the start codon of greater). The source of synthetic APF is from Peptides Inter CKAP4 and ended at the stop codon of YPF. national (Louisville, Ky.). The positive response is defined as HeLa cells (ATCCHCCL-2), (SCHERER W F, SYVER the rate and degree of translocation of the APF reporter TONJ T. GEY GO. Studies on the propagation in vitro of 10 CKAP4:YFP. Clonal cell lines that respond appropriately poliomyelitis viruses. IV. Viral multiplication in a stable (defined here as: the translocation of the reporter CKAP4: strain of human malignant epithelial cells (strain HeLa) YFP into the nucleus in one hour, to a degree sufficient to be derived from an epidermoid carcinoma of the cervix. J Exp statistically greater (defined here as two standard deviations Med. 1953 May: 97(5):695-710) a human cervical adenocar greater than the mean of control or more) than controls. cinoma cell line with epithelial cell-like characteristics were 15 Clonal lines that pass the positive-response test will be char obtained from the AmericanType Culture Collection (Ameri acterized further by assessing their response to varying doses can Type Culture Collection (ATCC) (P.O. Box 1549 Manas of synthetic APF ranging from 0, beginning at 10 fM and up sas, Va. 20108 USA). The cells were maintained in culture to 100 nM in 10-fold incremental increases in the dose of and subcultured according to the following protocol: ATCC synthetic APF. Clona cell lines that respond to the variable complete growth medium: Dulbecco's minimal essential dose in a fashion that fits well to a sigmoidal curve will be medium (DMEM). To make the complete growth medium, deemed “APF-reporter clonal cell lines”. add the following components to the base medium: fetal bovine serum to a final concentration of 10%. Atmosphere: Example 3 air, 95%: carbon dioxide (CO2), 5% Temperature: 37.0° C. Subculturing Protocol: 25 Characterization of Nuclear Translocation Assay Using 1. Remove and discard culture medium. Synthetic APF: To determine the dynamic range of this assay, 2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin we will first measure the degree of CKAP4:YFP nuclear 0.53 mM EDTA solution to remove all traces of serum translocation in response to varying concentrations of syn which contains trypsin inhibitor. thetic APF (Peptides International) ranging from 10 fM to 3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flaskand 30 100 nM (control—no APF) using the reporter cell line observe cells under an inverted microscope until cell described above. If the lowest concentration of APF stimu layer is dispersed (usually within 5 to 15 minutes). Note: lates translocation of CKAP4:YFP, it will be reduced further To avoid clumping do not agitate the cells by hitting or until no response is seen. This will allow us to define the lower shaking the flask while waiting for the cells to detach. end of sensitivity of the assay. For each APF concentration, Cells that are difficult to detach may be placed at 37°C. 35 the abundance of CKAP4:YFP in the nucleus will be mea to facilitate dispersal. Sured using morphometry and high-content screening algo 4. Add 6.0 to 8.0 ml of complete growth medium and rithms (e.g., NIS Elements, Nikon; CytoShop 2.0, Vala Sci aspirate cells by gently pipetting. ences, respectively) that can measure the abundance of a 5. Add appropriate aliquots of the cell Suspension to new targeted fluorophore at almost any desired location in the cell culture vessels. 40 (e.g., plasma membrane, nucleus, nucleolus) (Morelocket al. 6. Incubate cultures at 37° C. 2005; Mikic et al., 2006). The abundance of CKAP4:YFP fluorescence in the nucleus will be quantitated by taking two Example 2 separate images of the cells. The first image will be of DRAQ5 (a far-red, nucleic acid binding fluorophore that A Subcultivation Ratio: A Subcultivation ratio of 1:2 to 1:6 45 avoids emission overlap with YFP) staining in the nucleus; was used. Generation of cell lines that stably express the APF the second of CKAP4:YFP fluorescence. An algorithm will reporter CKAP4:YFP. HeLa cells stably expressing CKAP4: then quantify the amount of CKAP4:YFP fluorescence that is YFP will be generated by two standard methods. In the first coincident with the nuclear stain, and Subsequent, built-in method, HeLa cells will be transfected with a mammalian statistical analyses will be used to determine the variability expression plasmid, pcDNA3CKAP4:YFP using the 50 among cells to a single treatment. Statistical analyses of the FuGene6 reagent. This plasmid also encodes resistance to differences between the mean values for populations of cells geneticin and will be maintained with 0.4 mg/ml geneticin. treated with different concentrations will be determined by The second method will use lentiviral infection at a multiplic 2-way ANOVA analyses. The algorithm to quantify nuclear ity of infection equal to one integrant per cell-host genome. translocation is among the oldest, most robust morphometric, The cDNA expressing the reporter will be cloned into 55 high-content screening algorithms in existence. Using these pI entiG.3 TOPO (Invitrogen) or a similar vector. Clonal cell programs, large numbers of cells (100s to thousands in each lines stably expressing the APF reporter CKAP4:YFP will be field of view for each condition) can be measured to deter selected by exposure of infected cells to culture medium mine mean values for the nuclear translocation with a high containing 20 micrograms/mL blasticidin. Stable transfec degree of statistical confidence. These measurements will tants generated from each method will be chosen based on 60 provide a dose response curve from which the EC50 for similarity to endogenous CKAP4 distribution, sorted by synthetic APF-induced CKAP4:YFP translocation will be FACS (fluorescence activated cell sorter) to obtain a homo determined as well as the sensitivity of the assay. Our pre geneous population of cells expressing CKAP4:YFP, and liminary data indicated that CKAP4:YFP translocation maintained using standard cell culture techniques. A prelimi occurred within two hours of APF treatment; therefore, for nary evaluation using microscopic, visual examination of the 65 determining the dose-dependent response, we will make ini expression will be done to select clones that express the tial morphometric measurements 15 min, 30 min, 60 min, 90 reporter in a manner that resembles the endogenous pattern of min, 2 hr, 4 hr, and 6hr after APF application on fixed cells. US 9,075,045 B2 73 74 This time course can then be adjusted (to focus in on a win which each IC patient or control wipes the labial area with dow for maximal signal to noise ratio) and the dose-depen 10% povidone iodine solution and then collects midstream dent response remeasured after we make a more thorough urine into a sterile container. Specimens will be kept at 4°C. determination of the rate of CKAP4:YFP translocation as and then transported to our laboratory for removal of cellular described below. debris by low speed centrifugation at 4°C. Each sample will The rate of CKAP4:YFP translocation will be assessed by be aliquoted under sterile conditions and stored at -80° C. time-lapse imaging during exposure of HeLa cells to a satu until ready for use in the protocol listed below. Importantly, rating dose of APF (as determined in the dose response the same urine samples will be tested for APF activity using a experiments). CKAP4:YFP nuclear abundance will be mea cellular proliferation assay (3H-thymidine incorporation sured as described above and plotted over time. The rate (t/2) 10 measured by the method of Keay et al., 1996) in order to of CKAP4:YFP nuclear translocation and time to plateau will validate the effectiveness of the CKAP4:YFP reporter system be determined by fitting the data to an appropriate curve using against the current bioassay for APF detection in patient Microcal Origin 6.0. Knowing the rate of translocation fol urine. lowing exposure of cells to a saturating dose of APF will 1. The day before the assay is performed, CKAP:YFP allow us to define the upper end of the dynamic range of the 15 HeLa cells will be seeded in 35 mm imaging dishes at a translocation metric. The difference between these two mea density of 50,000 cells/dish in normal DMEM and incu Sures, the dose-response (dynamic range) as well as the time bated overnight at 37° C. and 5% CO (resulting in course of the translocation, will allow us to determine approximately 60% confluence the next day). whether the assay yields an acceptable Z-score (Zhang et al. 2. The following day, the medium will be replaced with 1999). This coefficient takes into account the signal dynamic serum-free DMEM, and the cells inspected under a range and the data variation associated with the signal mea microscope to ensure that they look healthy and uncon Surements; therefore, it assesses the overall quality or robust taminated, and that there is no uneven distribution of ness of the assay and, ultimately, its usefulness as a diagnostic cells around the edge of the plate. assay for IC. The Z-score is the standard by which virtually all 3. 4-6 hours later, urine specimens from patients with IC or high-throughput and high-content assays are measured. In 25 controls will be adjusted to pH 7.2 and 300 mOsm, addition, determining the time dependence of the dose-de filtered through a 0.2-lum pore filter, diluted 1:2 in pendent response of CKAP4:YFP nuclear translocation will DMEM (with only Glutamax and antibiotic/antimycotic provide the parameters necessary to develop a “fixed-end Solution) and applied to the cells. point assay'. In this format, APF is applied to the reporter 4. Cell plates will be incubated for a predetermined amount cells in culture for a predetermined amount of time at which 30 of time (determined in section 1.2) at 37° C. and 5% point the cells will be fixed, the nuclei stained, and the amount CO. of CKAP4:YFP fluorescence in the nucleus measured as 5. Cell medium will be removed and the cells will be fixed described above. This assay format is easily standardized and for 10 min at room temperature in 4% formaldehyde in therefore has the most potential for widespread, commercial phosphate buffer pH-7.0. SC. 35 6. Cells will be washed 4 times with phosphate buffered Validation of CKAP4:YFP Nuclear Translocation Assay saline (PBS) and then the cell nucleiwill be stained by adding Using Patient Urine: To validate this assay for further devel 500 ul 1 uM DRAQ5 (Biostatus) in PBS containing 1 mM opment as a clinical diagnostic test for IC, we will determine MgSO. its ability to report APF activity in urine of women clinically 7. The abundance of CKAP4:YFP in the nuclei will be diagnosed with IC (according to NIDDK criteria). Adult 40 measured as described above. The concentration of APF women comprise 90% of patients diagnosed with this dis in the patient’s urine will be determined by comparing ease; therefore, recruitment of female patients for this initial the degree of translocation to the dose-response curve feasibility study will be more straightforward to achieve. generated in experiments described above. Urine from asymptomatic women and from women diag nosed with acute bacterial cystitis will be included as con 45 Example 4 trols. Urine will be collected at Mercy Hospital from women with IC at least 3 months after the most recent known bacte Statistical Analysis: The mean and standard deviation of rial urinary tract infection and one month after the last anti the APF concentration measured in the urine from each biotic use. Specimens will be obtained from women during patient population (IC, bacterial cystitis, and asymptomatic) routine office visits to Advanced Gynecological Associates at 50 will be determined. These values will be subjected to a two Mercy Hospital for management of IC. Asymptomatic con way ANOVA analysis to determine whether or not they are trols will be female volunteers with no history of IC or other significantly different from each other. Importantly, similar urologic disease and will be required to have no symptoms of numbers of patients have been used to successfully determine urinary tract infection or antibiotic use for at least 1 month. the validity of cellular proliferation as a diagnostic assay for Urine will also be collected at the time of diagnosis from 55 IC (Keay et al., 1998). The variability that exists among the female patients with acute bacterial cystitis (diagnosis based members of a single population, in particular the IC patient on the presence of more than 103 bacteria/mL with a single population may provide information that allows us to further type of bacterium isolated plus pyuria in combination with correlate disease severity or length of time since diagnosis to appropriate symptoms). All participants will be at least 18 APF concentration. To examine Such a relationship, patients years old and enrolled in accordance with guidelines of the 60 in all categories will be binned according to the amount of Scranton Temple Residency Program Instructional Review APF that is present in their urine. These categories will then Board. Participants will also be required to read and sign an be analyzed with respect to clinically relevant factors includ informed consent form. We aim to recruit a minimum of 50 ing patient age, severity of IC, and length of time since diag patients from each category, as similar numbers of patients nosis (or when symptoms first appeared) and their conditional have been used to successfully determine the validity of cel 65 likelihood measured. Ultimately, these analyses are intended lular proliferation as a diagnostic assay for IC (Keay et al. to provide 1) an indication of whether to move forward with 1998). Urine will be collected by the clean catch method in a larger-scale clinical trial aimed at determining the utility of US 9,075,045 B2 75 76 this assay as a diagnostic test for IC in men and women and 2) Immunocytochemistry—HeLa cells stably transfected a first glimpse at the relationship between the clinical pathol with WT CKAP4 or CKAP4C100S were seeded at a density ogy and symptomology of IC and APF concentration in of 2x104 cells/well in 8-well LabTek chamber slides (Nalge patient urine. Nunc) and grown to semiconfluence in DMEM medium con DNA Constructs—A vector construct containing wild- 5 taining 10% FBS, 100 U/ml penicillin, 100 lug/ml streptomy type CKAP4 (WT CKAP4) fused in-frame to the N-terminus cin, 1 ug/ml fungizone, and 0.4 mg/ml G418 (all from Invit of the V5 and 6xHis epitope tags was generated by PCR using rogen). Cells were fixed for 20 minutes with 3% CKAP4 specific primers and cDNA from HeLa cells. A paraformaldehyde in PBS, permeabilized with 0.1% Triton palmitoylation-incompetent form of CKAP4 (CKAP4 X-100 in PBS, and blocked in PBS/5% NGS (normal goat C100S) was created using site-directed mutagenesis (Strat 10 serum). Cells transfected with DHHC2 siRNA and treated agene, LaJolla, Calif.) to alter the cysteine at position 100 to with synthetic APF (Peptides International, Louisville, Ky.) serine. DHHC2 was cloned in-frame to the C-terminus of were fixed using ethanol/acetone (1:1) for 15 minutes at room mCFP to generate an N-terminal CFP fusion protein. All temperature and washed three times with 1xPBS prior to constructs were verified by DNA sequencing. blocking in PBS/5% NGS. The following primary antibodies Cell Culture and Transfections—HelLa (ATCC #CCL-2: 15 were used: mouse mab G1/296 against CKAP4 (“anti American Type Culture Collection, Manassa, Va.) cells were CLIMP-63, diluted 1:100, Alexis Biochemicals, San Diego, maintained in Dulbecco's modified Eagle's medium Calif.), rabbit pAb against calreticulin (diluted 1:1000, (DMEM) containing 10% fetal bovine serum (FBS), 100 Abeam, Cambridge, Mass.), and fluorescein isothiocyanate U/ml penicillin, 100 g/ml streptomycin, and 1 fungizone (all (FITC)-conjugated mouse mab against the V5 epitope (di from Invitrogen). Cells were transfected using FuGENE6 luted 1:500, Invitrogen). Secondary antibodies were FITC reagent (Roche, Basel, Switzerland) according to the manu labeled goat anti-rabbit or goat anti-mouse (diluted 1:1000, facturers instructions. To obtain stable clones, cells were Invitrogen) and tetramethyl rhodamine isothiocyanate diluted into 96-well plates (100 cells/well) 24 hours post (TRITC)-labeled goat anti-mouse (diluted 1:1000, Jackson transfection and selected in the presence of 0.4 mg/ml Gene ImmunoResearch Laboratories, West Grove, Pa.). Slides ticin (G418) (Invitrogen, Carlsbad, Calif.). 25 were mounted in SlowFade Antifade reagent (Invitrogen) and Normal primary bladder (NB) epithelial cells were isolated imaged using a Nikon TE2000 epifluorescence microscope. from patients as previously described (Keay et al., 1996; Keay Quantitative Real-Time PCR (qRT-PCR) Total RNA was et al., 2000; Keay et al., 2004a: Conrads et al., 2006). Cells extracted from synthetic APF, inactive control peptide were propagated in DMEM-F12 (Media-Tech, Manassas, treated, or control untreated NB epithelial cells using the Va.) with 10% heat inactivated FBS, 1% antibiotic/antimy 30 RNeasy Plus Mini Kit (Qiagen, Valencia, Calif.) according to cotic solution, 1% L-glutamine, 0.25 U/ml insulin (Sigma, St. the manufacturer's protocol. RNA was assessed by visualiza Louis, Mo.), and 5 ng/ml human epidermal growth factor (R tion of the 28S/18S ribosomal RNA ratio on a 1% agarose gel, & D Systems, Minneapolis, Minn.) at 37° C. in a 5% CO2 and total RNA concentration determined by measuring the atmosphere, and characterized by binding of AE-1/AE-3 pan absorbance of each sample at 260 nm and 280 nm using a cytokeratin antibodies (Signet, Emeryville, Calif.) as previ 35 Gene Quant RNA/DNA Calculator (GE Healthcare, Piscat ously described (Keay tal., 1996; Keay et al., 2004b). away, N.J.). Quantitative RT-PCR for gene expression was siRNA-Double-stranded siRNA (ON-TARGETplusTM) performed using Quantitect Primers (Qiagen), SYBR Green targeting ZDHHC2 and nonsense siRNA (ONTARGET RT-PCR kit reagents (Qiagen), and a Roche System II Light plusTM Control siRNA) were purchased from Dharmacon Cycler (software version 3.5). Samples were tested in tripli (Lafayette, Colo.). HeLa cells were trypsinized for 5 minutes 40 cate runs, and specific mRNA levels quantified and compared at 37°C., centrifuged in DMEM growth medium, and the cell to mRNA levels for B-actin using real-time PCR analysis pellet was resuspended in serum-free medium at a density of software from Applied Biosystems (Foster City, Calif.). 1x106 cells/ml. 200LA of the cell suspension was then trans Western Blot Analysis—Cells were lysed in ice-cold RIPA ferred to a sterile 2 mm cuvette with 14 siRNA and electropo buffer containing protease inhibitors (Pierce, Rockford, Ill.), rated at 160 V/500 uF capacitance using a BioRad Gene 45 sonicated, and centrifuged for 15 minutes at 4°C. The super Pulser Xcell. The cells were immediately transferred to natant protein concentration was measured using a Folin 96-well plates for thymidine incorporation assay or to reagent-based protein assay kit (BioRad, Hercules, Calif.). LabTek multiwell glass slides (Nalge Nunc, Rochester, N.Y.) Proteins were separated by electrophoresis using 4-12% for immunocytochemistry. To determine the effectiveness of NuPAGE Novex Bis-Tris polyacrylamide gels in MOPS run siRNA-mediated knockdown we used quantitative real-time 50 ning buffer (Invitrogen) and then transferred to nitrocellulose. PCR to measure the abundance of ZDHHC2 mRNA at times Membranes were blocked for 2 hours at room temperature in 0, 12, 24, 48, 72 and 96 hours following transfection of the TBST buffer (Tris-buffered saline, pH 7.4, with 0.1% Tween siRNA. These experiments were run in triplicate. 20) containing 5% nonfat milk and incubated with specific 3H-Thymidine Incorporation Cell proliferation was antibodies against vimentin (diluted 1:2000; BD Pharmin measured by 3H-thymidine incorporation into the DNA of 55 gen, San Jose, Calif.) or ZO-1 (diluted 1:125; Zymed Labo HeLa or NB epithelial cells. Briefly, synthetic APF or inactive ratories, San Francisco, Calif.) overnight at 4°C. The mem control peptide (NeoMPS, San Diego, Calif.) was resus branes were subsequently washed with TBST, incubated for 1 pended in acetonitrile/distilled water (1:1), diluted in serum hour at room temperature in HRPconjugated goat anti-mouse free DMEM, and applied to HeLa or NB cells; cell controls (diluted 1:40000; Santa Cruz, Biotechnology, Santa Cruz, received acetonitrile? distilled water diluted in serum-free 60 Calif.) or goat antirabbit (diluted 1:10000; Pierce) secondary DMEM alone. Cells were then incubated at 37° C. in a 5% antibodies, and developed by enhanced chemiluminescence CO2 atmosphere for 48 hours. The cell contents were har (Pierce). To assess equal loading of protein, the membranes vested and methanol-fixed onto glass fiber filterpaper, and the were stripped and reprobed for B-actin (diluted 1:5000; amount of radioactivity incorporated determined. Significant Sigma). The membranes were exposed to film (BioMax AR, inhibition of 3H-thymidine incorporation was defined as a 65 Kodak, Rochester, N.Y.) and the resulting images scanned at mean decrease in counts perminute of greater than 2 standard 300 dpi. The protein bands of interest were quantified using deviations from the mean of control cells for each plate. Image.J and the integrated signal densities normalized first to US 9,075,045 B2 77 78 B-actin (the loading control) and Subsequently expressed in on a root Scale, which is an intermediate between log and terms of the fractional abundance relative to untreated control linear scales. The Affymetrix MASS algorithm was used for cells. array processing. Immunoprecipitation HeLa cells were transfected with ** Reference: Su AI, Wiltshire T, Batalov S. Lapp H, Ching mCFP:DHHC2 or mock-transferred using FuGENE6 reagent KA, Block D, Zhang J, Soden R. Hayakawa M. Kreiman G, (Roche) according to the manufacturers instructions. Forty Cooke MP. Walker JR, Hogenesch J B (2004) A gene atlas of eight hours later, cells were washed in PBS, lysed in 500 uL the mouse and human protein-encoding transcriptomes. Proc ice-cold lysis buffer (25 mM HEPES, 150 mM NaCl, 5 mM Natl AcadSci USA. 2004 Apr. 20; 101 (16):6062-7. MgCl, 1% NP-40, and protease inhibitor cocktail), and incu UniGene—electronic Northern Normal/eNorthern Can bated on ice for 90 minutes with Vortexing. After normalizing 10 cer. Electronic Northern: For the shown set of non-fetal nor for equal protein concentration, lysates were immunoprecipi mal and cancer human tissues, NCBI's Unigene dataset (Hs .data) is mined for information about the number of unique tated with a mAb against GFP (JL-8 BD Biosciences, San clones per gene per tissue. Clones are assigned to particular Jose, Calif.; also recognizes the GFP spectral mutant, CFP) tissues by applying data-mining heuristics to Unigene's overnight at 4°C. with rocking. Protein G Sepharose 4B 15 library information file (Hs.lib.info). Electronic expression (Invitrogen) was added the following day, and the samples results were calculated by dividing the number of clones per were incubated for an additional 18 hours at 4°C. with rock gene by the number of clones per tissue. They were then ing. Proteins in the immunoprecipitation complex were normalized by multiplying by 1M, and the obtained normal washed four times in ice-cold lysis buffer and then heated in ized counts are presented on the same root Scale as the experi SDS sample buffer before separation by SDS-PAGE and mental tissue vectors. transfer to nitrocellulose. Western blot analysis was per CGAP:SAGE Normal/SAGE Cancer formed as described above using a mAb against CKAP4 Serial Analysis of Gene Expression: For the same set of (G1/296, diluted 1:1000; Alexis Biochemicals). normal and cancer human tissues, CGAP datasets Hs.fre Wound-healing Migration Assay—HeLa cells stably trans quencies and Hs.libraries are mined for information about the fected with CKAP4WT, CKAP4C100S, or parental controls 25 number of SAGE tags per tissue. Tags are reassigned to a were seeded at a density of 4x104 cells/well in six-well plates Unigene cluster and after that to a particular gene by mining coated with fibronectin. Once the cells reached confluency, Hs.best gene, HS.best tag and Hs GeneData. The expres the culture medium was replaced with medium containing sion level of a particular gene in a particular tissue was cal AraC (5 lug/ml. Sigma) to block further cell division and culated as the number of appearances of the corresponding permit measurement of migration in the absence of prolifera 30 tag divided by the total number of tags in libraries derived tion. The scratch wound was made using a p200 pipette tip from that tissue. These fractions were then normalized by eight hours after the addition of AraC. Cell migration was multiplying by 1.2M and the obtained normalized counts are monitored Over a 24 hour period starting at the time the presented on the same root scale as that used for the electronic scratch wound was made and at three-hour intervals thereaf Northern pictures. Please note: Currently, only associations 35 with minimal ambiguity participate in the analysis. ter using a Nikon Eclipse TE2000-U microscope under 20x Source www.genecards.org. magnification and phase contrast. The percent of the wound remaining opentSEM was measured at the indicated time Example 6 points and calculated using Image J, with the 0 hour time point being set to 100%. The percentage of surface not cov 40 A Prophetic Example. Use of Dose-Response Curve gen ered by the cells was plotted versus time, and the data were fit erated with Synthetic APF. It is envisioned that CKAP4 to a monoexponential decay curve using Microcal Origin 6.0 nuclear translocation can be used to determine the concentra to determine the rate at which the cells migrated to fill the tion of APF in the urine of patients with IC. For the assay to be exposed area. The distance between the two migrating front useful in this capacity first it will be necessary to determine lines of cells was measured using the micrometer tool in NIS 45 the dynamic range of this assay by measuring the degree of Elements (Nikon, Melville, N.Y.), and the rate of migration CKAP4:YFP nuclear translocation in response to varying was determined and expressed as uM/hr. concentrations of synthetic APF ranging from 10 fM to 100 nM (control—no APF) using the reporter cell line described Example 5 above or endogenously expressed CKAP4. If the lowest con 50 centration of APF stimulates translocation of CKAP4:YFP, it Expression analyses of CKAP4 in a broad array of tissues will be reduced further until no response is seen. This will and disease states, FIG. 18. GeneNote/GNF Normal/GNF provide the lower end of sensitivity of the assay. For each APF Cancer Expression array images Experimental tissue vectors: concentration, the abundance of CKAP4:YFP in the nucleus Duplicate measurements were obtained for twelve normal can be measured using morphometry and high-content human tissues (out of 28 tissues shown) hybridized against 55 screening algorithms available on the ArrayScanCVTI HCS Affymetrix GeneChips HG-U95A-E (GeneNote data)and for Reader (e.g., VHCS Discovery ToolBox Software) that can 22 normal human tissues hybridized against HG-U133A measure the abundance of a targeted fluorophore at almost (GNF Symatlas data **). The intensity values (shown on the any desired location in the cell (e.g., plasma membrane, y-axis) were first averaged between duplicates, then probeset nucleus, nucleolus) (Morelocket al., 2005; Mikic et al., 2006). values were averaged per gene, global median-normalized 60 The abundance of CKAP4:YFP fluorescence or endogenous and scaled to have the same median of about 70 (half-way CKAP4 in the nucleus will be quantitated from two indepen between GeneNOte and GNF medians). Available at GNF dent images of the same cells. The first image will be of Symatlas, HG-U133A expression data for 18 NCI60 cancer DRAQ5 (a far-red, nucleic acid-binding fluorophore that cell lines was processed and added to the display (a single avoids emission overlap with YFP) staining in the nucleus (or measurement taken; normalized according to the GNF 65 a similar fluorescent, DNA-labeling compound); the second Symatlas normal data). The correspondence between cell of CKAP4:YFP fluorescence or immunolabeled, endogenous lines and tissues is shown. Normalized intensities are drawn CKAP4. An algorithm will quantify the amount of CKAP4: US 9,075,045 B2 79 80 YFP fluorescence or endogenous CKAP4 that is coincident multiple trials to determine the timepoint of maximum trans with the nuclear stain, and Subsequent, built-in statistical location. This fixed endpoint will be the standard time for all analyses will determine the variability among cells to a single Subsequent assays using patient urine. The degree of CKAP4:YFP or endogenous CKAP4 trans treatment. Statistical analyses of the differences between the location that is induced by the application of patient urine (IC mean values for populations of cells treated with different patient or otherwise) will be compared to the dose response concentrations will be determined by 2-way ANOVA analy curve obtained using synthetic APF. This standard method ses with age and treatment groups as the covariates. The will provide a determination of the concentration of APF in algorithm to quantify nuclear translocation is among the old the patient urine. Correlating the APF concentration mea est, most robust morphometric, high-content Screening algo sured with this assay to the clinical state of the patient will rithms. Using these programs, large numbers of cells (100s to 10 provide valuable insight into the onset, progression and thousands in each field of view for each condition) can be pathogenesis of IC especially when multiple measurements measured to determine mean values for the nuclear translo are made from individuals over time. cation with a high degree of Statistical confidence. These While the invention has been described in detail and with measurements will provide a dose response curve from which 15 reference to specific examples thereof, it will be apparent to the EC50 for synthetic APF-induced CKAP4:YFP or endog one skilled in the art that various changes and modifications enous CKAP4 translocation will be known. The measure can be made therein without departing from the spirit and ments will be made at a fixed timepoint as determined by Scope thereof.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 22

SEO ID NO 1 LENGTH: 31 TYPE: DNA ORGANISM: Artificial FEATURE; OTHER INFORMATION: Primer

<4 OOs SEQUENCE: 1

Caccggit acc atgcc ct cqg ccaaacaaag g 31

SEO ID NO 2 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial FEATURE; OTHER INFORMATION: Primer

<4 OOs SEQUENCE: 2

aattcticag gaccttitt.cg tdaatctitct coactitt cac 4 O

SEO ID NO 3 LENGTH: 30 TYPE: DNA ORGANISM: Artificial FEATURE; OTHER INFORMATION: Primer

<4 OOs SEQUENCE: 3

tatt ct cag atggtgagca agggcgagga 3O

SEO ID NO 4 LENGTH: 30 TYPE: DNA ORGANISM: Artificial FEATURE; OTHER INFORMATION: Primer

<4 OOs SEQUENCE: 4

citcggaattic titacttgtac agct cqtcca 3O

<21 Os SEQ ID NO 5 &211s LENGTH: 2913 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

US 9,075,045 B2 83 84 - Continued gagatat citc t cacttgtct tcc actittga accggittaaa gct catagct gtcagctctg 24 OO aatgaggagg ggagaa.gc.cc ctgggtctitt Ctttgaaagg aatcc.gctgc ttgagggctg 246 O cotic cct cat togttctott cctgacgcat ctgtgatatic agaggtaact 252O atgcaaag.ca tcc aggcggit tctgaatgtg aag cactaca CCC agcagag tcc.cggtgcc 2580 citctgtc.ccc actgc.cggcc catgtcct ct Ctc.cggaggit Caccalaggaa tgcacaggtt 264 O tcqactacca gaaaggggag tcc ttgggitt Ctttcaaaaa. attctgagg agagctgtct 27 OO acagtggaat aggggg.tctic Cctggggaat gCaggccaag t cottt tatt tta acatgat 276 O gtcCatgaag aggtttgc.cg tctgggcagc Cctgtcggca aggagcgtgc at actg.cgitt 282O tgtgtaattg tittgctgtat ctic cottic cc tctgagctgt attgttctitt aatggctgtc. 288O ttgccct tcc aaaaaaaatt gaaaaaaaaa a.a.a. 291.3

<210s, SEQ ID NO 6 &211s LENGTH: 6O2 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Met Pro Ser Ala Lys Glin Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1. 1O 15

Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Wall 25 3O

Ala Lys Pro Pro Pro Ala Pro Glin Gln Pro Pro Pro Pro Pro Ala 35 4 O 45

Pro Pro Glin Gln His Pro Glin Gln His Pro Glin Asn Glin Ala His 55 6 O

Gly Gly Gly His Arg Gly Gly Gly Gly Ser Ser 65 70 8O

Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser 85 90 95

Ser Ala Ser Cys Ser Arg Arg Lieu Gly Arg Ala Lell Asn Phe Lieu. Phe 11 O

Luell Ala Lieu Wall Ala Ala Ala Ala Phe Ser Gly Trp Wall His 115 12 O 125

His Wall Luell Glu Glu Wall Glin Glin Val Arg Arg Ser His Glin Asp Phe 13 O 135 14 O

Ser Arg Glin Arg Glu Glu Lieu. Gly Glin Gly Lieu. Glin Gly Wall Glu Glin 145 150 155 160

Wall Glin Ser Leul Glin Ala Thr Phe Gly Thr Phe Glu Ser Ile Lieu. 1.65 17O 17s

Arg Ser Ser Gln His Lys Glin Asp Lieu. Thir Glu Ala Wall Lys Glin 18O 185 19 O

Gly Glu Ser Glu Val Ser Arg Ile Ser Glu Wall Lell Glin Lieu. Glin 195

Asn Glu Ile Lieu Lys Asp Lieu. Ser Asp Gly Ile His Wall Wall 21 O 215 22O

Ala Arg Glu Arg Asp Phe Thr Ser Lieu. Glu Asn Thir Wall Glu Glu Arg 225 23 O 235 24 O

Lell Thir Glu Lieu. Thir Lys Ser Ile Asn Asp Asn Ile Ala Ile Phe Thr 245 250 255

Glu Wall Glin Lys Arg Ser Gln Lys Glu Ile ASn Asp Met Lys Ala Lys 26 O 265 27 O US 9,075,045 B2 85 86 - Continued

Wall Ala Ser Lieu. Glu Glu Ser Glu Gly Asn Lys Glin Asp Luell Lys Ala 27s 28O 285

Lell Lys Glu Ala Wall Lys Glu Ile Glin Thir Ser Ala Lys Ser Arg Glu 29 O 295 3 OO

Trp Asp Met Glu Ala Lieu. Arg Ser Thr Lieu. Glin Thir Met Glu Ser Asp 3. OS 310 315

Ile Tyr Thr Glu Val Arg Glu Lieu Val Ser Lieu. Glin Glu Glin Glin 3.25 330 335

Ala Phe Lys Glu Ala Ala Asp Thr Glu Arg Lieu. Ala Lell Glin Ala Luell 34 O 345 35. O

Thir Glu Lys Lieu. Lieu. Arg Ser Glu Glu Ser Val Ser Arg Luell Pro Glu 355 360 365

Glu Ile Arg Arg Lieu. Glu Glu Glu Lieu. Arg Glin Lell Ser Asp Ser 37 O 375

His Gly Pro Lys Glu Asp Gly Gly Phe Arg His Ser Glu Ala Phe Glu 385 390 395 4 OO

Ala Lieu. Glin Glin Llys Ser Glin Gly Lieu. Asp Ser Arg Lell Glin His Wall 4 OS 41O 415

Glu Asp Gly Val Lieu. Ser Met Glin Val Ala Ser Ala Arg Glin Thir Glu 42O 425 43 O

Ser Lieu. Glu Ser Lieu Lleu Ser Lys Ser Glin Glu His Glu Glin Arg Luell 435 44 O 445

Ala Ala Lieu. Glin Gly Arg Lieu. Glu Gly Lieu. Gly Ser Ser Glu Ala Asp 450 45.5 460

Glin Asp Gly Lieu Ala Ser Thr Val Arg Ser Lieu. Gly Glu Thir Glin Luell 465 470 47s

Wall Lieu. Tyr Gly Asp Val Glu Glu Lieu Lys Arg Ser Wall Gly Glu Luell 485 490 495

Pro Ser Thr Val Glu Ser Leu Gln Llys Val Glin Glu Glin Wall His Thir SOO 505

Lell Lieu. Ser Glin Asp Glin Ala Glin Ala Ala Arg Lell Pro Pro Glin Asp 515 52O 525

Phe Lieu. Asp Arg Lieu. Ser Ser Lieu. Asp Asn Lieu. Lys Ala Ser Wall Ser 53 O 535 54 O

Glin Val Glu Ala Asp Lieu Lys Met Lieu. Arg Thr Ala Wall Asp Ser Luell 5.45 550 555 560

Wall Ala Tyr Ser Val Lys Ile Glu Thir Asn. Glu Asn Asn Luell Glu Ser 565 st O sts

Ala Lys Gly Lieu. Lieu. Asp Asp Lieu. Arg Asn Asp Lell Asp Arg Luell Phe 58O 585 59 O

Wall Llys Val Glu Lys Ile His Glu Lys Val 595 6OO

<210s, SEQ ID NO 7 &211s LENGTH: 4 O12 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 gtgggagcCC gC9aggtggC cc.gagcgagg gCCCC gcatc cc.cgcagagc cc.gc.ccgagg 6 O gcgcagcctg. CCaC9C Cagg gagg.cggcc ggg.cggcggg galacggcggg gC9gagcgc 12 O agcct c ccga cqc cqc cqcc ticaccgcc cc cct cogcctic Ctcggcct cc gct cqcago C 18O gcc.gc.ctic.cg cct cogcc.gg gctgaggagc cqggagt cc.g 24 O

US 9,075,045 B2 89 - Continued ttagtttgag ataaactaaa gtgtgttgat gcc agaatgt to agctt cag taaatataat 264 O aagct cittgt gcc titg tatg cactatttaa aaaaagttitt ttittatttga gtc.cagtata 27 OO att catgitaa atgttaacaa ttagaataat actictd tatgcttttittgat actgattittg 276 O agaatttaaa gcagattacc ttittaaaact ggaccaacta agtaattggit atttaat caa 282O agagaaaatg gtaataaact tttcaaaatc tttgttaaac caaac attca acacaaaata 288O aact agaagg ccagaggata atggaataaa agat cattgc aattactitat cottcctaaa 294 O aatatagttt tat attaatt gtgct tatgg aagaaacaat gtcagccaag to catttitat 3 OOO agtttgagtg caattctittgaacaatagaa at atctgcag tottt cacag atttgtatta 3 O 6 O tgctgaagag titt catctga caatctgctt caagaaatct cagaaaatat gataa cattt 312 O taactitt cat tittagagcac gttittggit catttittaaaaa tacctaaagt gccagac cog 318O aacctatago tactgctaga agt cittaaaa aaaccaa.cag cagcacagga tigt attalaga 324 O attatatgaa gtcaggtttgtttitttttitt tttittttittt toaaag.caca gtactgttag 33 OO Ctgtttttgt gga caggatt Cattaagta t t c cct ctitg tcaaactgga agctagggga 3360 aaaagaggga tttittatcct ttact cittct agagtactgt taatgcc cct titc.ccacagt 342O cittittatata attaaatata tdt caataca cattagaatc agatttgaaa aagttaaaac 3480 aattt cattgttgtaattgt tocctttctg ttitt catata gtgaataacc tittaaagggit 354 O tgttttgttt tdttittgaat tataggagitt ataatctittg gagatgattg catat ct cat 36OO tagatatgca atataaattt atctgagtga acaaagtgct aaataaatag atctacattt 366 O tgtacatatt tatataaaat ttacctittaa gtatt tactt taaaaaattt aatggct taa 3.720 citcgaactitg aagacacata cittcaactgt cct tattgtc. cattaaact g ataattittga 378 O tttittcttgc titttatagat titt actatat aggaatcaag atttaagaaa ttittgcatta 384 O aaaatagtgt accaatgctt catatacgitt agittatttgc tattatgtag gigaagaggat 3900 tgttattt ca aagatatatt aaagaacagt togcatctgaa tataatcatg atgcattcaa 396 O tgaagttcat atc catgaat t cact cotaa tataccctaa taaagtggitt ga 4 O12

<210s, SEQ ID NO 8 &211s LENGTH: 367 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 Met Ala Pro Ser Gly Pro Gly Ser Ser Ala Arg Arg Arg Cys Arg Arg 1. 5 1O 15 Val Lieu. Tyr Trp Ile Pro Val Val Phe Ile Thr Lieu Lleu Lieu. Gly Trp 2O 25 3O Ser Tyr Tyr Ala Tyr Ala Ile Glin Lieu. Cys Ile Val Ser Met Glu Asn 35 4 O 45 Thr Gly Glu Glin Val Val Cys Lieu Met Ala Tyr His Lieu. Leu Phe Ala SO 55 6 O

Met Phe Val Trp Ser Tyr Trp Llys Thr Ile Phe Thr Lieu Pro Met Asn 65 70 7s 8O

Pro Ser Lys Glu Phe His Lieu. Ser Tyr Ala Glu Lys Asp Lieu. Lieu. Glu 85 90 95 Arg Glu Pro Arg Gly Glu Ala His Glin Glu Val Lieu. Arg Arg Ala Ala 1OO 105 11 O

Lys Asp Leu Pro Ile Tyr Thr Arg Thr Met Ser Gly Ala Ile Arg Tyr 115 12 O 125 US 9,075,045 B2 91 92 - Continued

Cys Glin Lieu. Ile Llys Pro Asp Arg Cys His His Cys Ser 13 O 135 14 O

Val Cys Asp Lys Cys Ile Lieu Lys Met Asp His His Pro Trp Val 145 150 155 160

Asn Asn. Cys Val Gly Phe Ser Asn Tyr Llys Phe Phe Lell Luell Phe Lieu. 1.65 17O 17s

Ala Tyr Ser Lieu. Lieu. Tyr Cys Lieu Phe Ile Ala Ala Thir Asp Lieu. Glin 185 19 O

Tyr Phe Ile Llys Phe Trp Thr Asn Gly Leu Pro Asp Thir Glin Ala Lys 195

Phe His Ile Met Phe Leu Phe Phe Ala Ala Ala Met Phe Ser Wall Ser 21 O 215

Leul Ser Ser Leu Phe Gly Tyr His Cys Trp Lieu. Wall Ser Asn Lys 225 23 O 235 24 O

Ser Thir Lieu. Glu Ala Phe Arg Ser Pro Wall Phe Arg His Gly Thir Asp 245 250 255

Lys Asn Gly Phe Ser Lieu. Gly Phe Ser Lys Asn Met Arg Glin Wall Phe 26 O 265 27 O

Gly Asp Glu Llys Llys Tyr Trp Lieu Lieu Pro Ile Phe Ser Ser Lieu. Gly 285

Asp Gly Cys Ser Phe Pro Thr Cys Lieu Wall Asn Glin Asp Pro Glu Glin 29 O 295 3 OO

Ala Ser Thr Pro Ala Gly Lieu. Asn Ser Thir Ala Asn Luell Glu Asn 3. OS 310 315 32O

His Glin Phe Pro Ala Lys Pro Lieu Arg Glu Ser Glin Ser His Lieu. Luell 3.25 330 335

Thr Asp Ser Glin Ser Trp Thr Glu Ser Ser Ile Asn Pro Gly 34 O 345 35. O

Lys Ala Gly Met Ser Asn. Pro Ala Lieu. Thir Met Glu Asn Glu Thir 355 360 365

<210s, SEQ I D NO 9 &211s LENGT H: 74. O 212. TYPE : DNA <213> ORGANISM: Artificial 22 Os. FEATU RE: <223> OTHER INFORMATION: Artifical Construct

<4 OOs, SEQUENCE: 9 atggtgagca agggcgagga gctgttcacc ggggtggtgc c catcctggit cgagctggac 6 O ggcgacgtaa acggcc acaa gttcagogtg tCcgg.cgagg gcgaggg.cga tgccaccitac 12 O ggcaa.gctga c cctdaagct gatctgcacc accggcaa.gc ctgg.cccacc 18O citcgtgacca CCCtgggcta cggcctgcag gct accc.cga C Cacatgaag 24 O

Cagcacgact tottcaagtic cgc catgc cc gaaggctacg tccaggagcg CaCCatctt C 3OO ttcaaggacg acggcaacta Caagaccc.gc gcc.gaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaa.cat Cctggggcac aagctggagt acalactacala cagccacaac gtctatat ca cc.gc.cgacaa gCagaagaac 48O ggcatcaagg CCaact tcaa. gatcc.gc.cac alacatcgagg acggcggcgt gcagotcgc.c 54 O gaccact acc agcagaacac c cc catcggc gacggcc.ccg cgacaac cac tacctgagct accagt ccaa gctgagcaaa gaccc.caacg agaag.cgcga t cacatggit c 660

Ctgctggagt tcgtgaccgc cgc.cgggatc act ct cqgca tggacgagct gtacaagtaa 72 O US 9,075,045 B2 93 - Continued ttacttgtac agct cqtcca 74 O

<210s, SEQ ID NO 10 &211s LENGTH: 239 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artifical Construct

<4 OOs, SEQUENCE: 10 Met Val Ser Lys Gly Glu Glu Lieu Phe Thr Gly Val Val Pro Ile Leu 1. 5 1O 15 Val Glu Lieu. Asp Gly Asp Val Asn Gly His Llys Phe Ser Val Ser Gly 2O 25 3O Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Lieu. Thir Lieu Lys Lieu. Ile 35 4 O 45 Cys Thir Thr Gly Lys Lieu Pro Val Pro Trp Pro Thr Lieu Val Thir Thr SO 55 6 O Lieu. Gly Tyr Gly Lieu. Glin Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 7s 8O Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Glin Glu 85 90 95 Arg Thir Ile Phe Phe Lys Asp Asp Gly Asn Tyr Llys Thr Arg Ala Glu 1OO 105 11 O Val Llys Phe Glu Gly Asp Thir Lieu Val Asn Arg Ile Glu Lieu Lys Gly 115 12 O 125 Ile Asp Phe Lys Glu Asp Gly Asn. Ile Lieu. Gly His Llys Lieu. Glu Tyr 13 O 135 14 O Asn Tyr Asn. Ser His Asn Val Tyr Ile Thr Ala Asp Llys Glin Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn. Phe Lys Ile Arg His Asn. Ile Glu Asp Gly Gly 1.65 17O 17s Val Glin Lieu Ala Asp His Tyr Glin Glin Asn Thr Pro Ile Gly Asp Gly 18O 185 19 O Pro Val Lieu. Lieu Pro Asp Asn His Tyr Lieu. Ser Tyr Glin Ser Lys Lieu. 195 2OO 2O5 Ser Lys Asp Pro Asn. Glu Lys Arg Asp His Met Val Lieu. Lieu. Glu Phe 21 O 215 22O Val Thir Ala Ala Gly Ile Thr Lieu. Gly Met Asp Glu Lieu. Tyr Lys 225 23 O 235

<210s, SEQ ID NO 11 &211s LENGTH: 591 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 11 Ala Arg Ser Pro Pro Ser Pro Arg Pro Glu Pro Pro Pro Arg Pro Pro 1. 5 1O 15

Cys Pro Arg Pro Asn Lys Gly Ala Pro Arg Ala Ala Thr Ala Pro Arg 2O 25 3O

Ala Pro Arg Arg Arg Val Pro Thr Arg Arg Ala Ala Arg Met Thir Trip 35 4 O 45

Arg Arg Ser Arg Arg Arg Arg Arg Ser Ser Arg Arg Arg Arg Pro Arg SO 55 6 O

Arg Thr Arg Ser Ser Thr Arg Ser Ser Thr Arg Arg Thr Arg Arg Thr US 9,075,045 B2 95 96 - Continued

65 70

Ala Arg Ala Ala Thir Ala Ala Ala Ala Ala Ala Ala Lell Gly Arg Ala 85 90 95

Lell Asn Phe Luell Phe Lell Ala Luell Wall Ala Ala Ala Ala Phe Ser 105 11 O

Gly Trp Cys Wall His His Wall Luell Glu Glu Wall Glin Glin Wall Arg Arg 115 12 O 125

Ser His Glin Asp Phe Ser Arg Glin Arg Glu Glu Lell Gly Glin Gly Luell 13 O 135 14 O

Glin Gly Wall Glu Glin Lys Wall Glin Ser Luell Glin Ala Thir Phe Gly Thir 145 150 155 160

Phe Glu Ser Ile Lell Arg Ser Ser Glin His Lys Glin Asp Luell Thir Glu 1.65 17s

Ala Wall Lys Glin Gly Glu Ser Glu Wall Ser Arg Ile Ser Glu Wall 18O 185 19 O

Lell Glin Lys Luell Glin Asn Glu Ile Luell Lys Asp Lell Ser Asp Gly Ile 195

His Wall Wall Asp Ala Arg Glu Arg Asp Phe Thir Ser Luell Glu Asn 21 O 215 22O

Thir Wall Glu Glu Arg Lell Thir Glu Luell Thir Lys Ser Ile Asn Asp Asn 225 23 O 235 24 O

Ile Ala Ile Phe Thir Glu Wall Glin Arg Ser Glin Glu Ile Asn 245 250 255

Asp Met Ala Lys Wall Ala Ser Luell Glu Glu Ser Glu Gly Asn 26 O 265 27 O

Glin Asp Luell Ala Lell Glu Ala Wall Glu Ile Glin Thir Ser 28O 285

Ala Lys Ser Arg Glu Trp Asp Met Glu Ala Luell Arg Ser Thir Luell Glin 29 O 295 3 OO

Thir Met Glu Ser Asp Ile Thir Glu Wall Arg Glu Lell Wall Ser Luell 3. OS 310 315

Glin Glu Glin Glin Ala Phe Glu Ala Ala Asp Thir Glu Arg Luell 3.25 330 335

Ala Luell Glin Ala Lell Thir Glu Luell Luell Arg Ser Glu Glu Ser Wall 34 O 345 35. O

Ser Arg Luell Pro Glu Glu Ile Arg Arg Luell Glu Glu Glu Luell Arg Glin 355 360 365

Lell Lys Ser Asp Ser His Gly Pro Glu Asp Gly Gly Phe Arg His 37 O 375

Ser Glu Ala Phe Glu Ala Lell Glin Glin Lys Ser Glin Gly Luell Asp Ser 385 390 395 4 OO

Arg Luell Glin His Wall Glu Asp Gly Wall Luell Ser Met Glin Wall Ala Ser 4 OS 415

Ala Arg Glin Thir Glu Ser Lell Glu Ser Luell Luell Ser Ser Glin Glu 425 43 O

His Glu Glin Arg Lell Ala Ala Luell Glin Gly Arg Lell Glu Gly Luell Gly 435 44 O 445

Ser Ser Glu Ala Asp Glin Asp Gly Luell Ala Ser Thir Wall Arg Ser Luell 450 45.5 460

Gly Glu Thir Glin Lell Wall Lell Gly Asp Wall Glu Glu Luell Arg 465 470

Ser Wall Gly Glu Lell Pro Ser Thir Wall Glu Ser Lell Glin Wall Glin 485 490 495 US 9,075,045 B2 97 - Continued

Glu Glin Val His Thr Lieu Lleu Ser Glin Asp Glin Ala Glin Ala Ala Arg SOO 505 51O Lieu Pro Pro Glin Asp Phe Lieu. Asp Arg Lieu. Ser Ser Lieu. Asp Asn Lieu. 515 52O 525 Lys Ala Ser Val Ser Glin Val Glu Ala Asp Lieu Lys Met Lieu. Arg Thr 53 O 535 54 O Ala Val Asp Ser Lieu Val Ala Tyr Ser Val Lys Ile Glu Thir Asn. Glu 5.45 550 555 560 Asn Asn Lieu. Glu Ser Ala Lys Gly Lieu. Lieu. Asp Asp Lieu. Arg Asn Asp 565 st O sts Lieu. Asp Arg Lieu. Phe Val Llys Val Glu Lys Ile His Glu Lys Val 58O 585 59 O

<210s, SEQ ID NO 12 &211s LENGTH: 526 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 12 Met Pro Ser Ala Lys Glin Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1. 5 1O 15 Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Gly 2O 25 3O Ala Glin Lieu. Ser Lieu. Lieu Pro Arg Pro Ala Ala Ala Ala Phe Ser Gly 35 4 O 45 Trp. CyS Val His His Val Lieu. Glu Glu Val Glin Glin Val Arg Arg Ser SO 55 6 O His Glin Asp Phe Ser Arg Glin Arg Glu Glu Lieu. Gly Glin Gly Lieu. Glin 65 70 7s 8O Gly Val Glu Gln Lys Val Glin Ser Leu Glin Ala Thr Phe Gly Thr Phe 85 90 95 Glu Ser Ile Lieu. Arg Ser Ser Glin His Lys Glin Asp Lieu. Thr Glu Lys 1OO 105 11 O Ala Wall Lys Glin Gly Glu Ser Glu Val Ser Arg Ile Ser Glu Val Lieu 115 12 O 125 Glin Llys Lieu. Glin Asn. Glu Ile Lieu Lys Asp Lieu. Ser Asp Gly Ile His 13 O 135 14 O Val Val Lys Asp Ala Arg Glu Arg Asp Phe Thir Ser Lieu. Glu Asn Thr 145 150 155 160 Val Glu Glu Arg Lieu. Thr Glu Lieu. Thir Lys Ser Ile Asn Asp Asn. Ile 1.65 17O 17s Ala Ile Phe Thr Glu Val Glin Lys Arg Ser Glin Lys Glu Ile Asn Asp 18O 185 19 O Met Lys Ala Lys Val Ala Ser Lieu. Glu Glu Ser Glu Gly Asn Lys Glin 195 2OO 2O5

Asp Lieu Lys Ala Lieu Lys Glu Ala Wall Lys Glu Ile Glin Thir Ser Ala 21 O 215 22O

Llys Ser Arg Glu Trp Asp Met Glu Ala Lieu. Arg Ser Thr Lieu. Glin Thr 225 23 O 235 24 O

Met Glu Ser Asp Ile Tyr Thr Glu Val Arg Glu Lieu Val Ser Lieu Lys 245 250 255

Glin Glu Glin Glin Ala Phe Lys Glu Ala Ala Asp Thr Glu Arg Lieu Ala 26 O 265 27 O

Lieu. Glin Ala Lieu. Thr Glu Lys Lieu. Lieu. Arg Ser Glu Glu Ser Val Ser US 9,075,045 B2 99 100 - Continued

28O 285

Arg Luell Pro Glu Glu Ile Arg Arg Luell Glu Glu Glu Lell Arg Glin Luell 29 O 295 3 OO

Lys Ser Asp Ser His Gly Pro Llys Glu Asp Gly Gly Phe Arg His Ser 3. OS 310 315

Glu Ala Phe Glu Ala Lieu. Glin Glin Ser Glin Gly Lell Asp Ser Arg 3.25 330 335

Lell Glin His Wall Glu Asp Gly Val Luell Ser Met Glin Wall Ala Ser Ala 34 O 345 35. O

Arg Glin Thir Glu Ser Lieu. Glu Ser Luell Luell Ser Ser Glin Glu His 355 360 365

Glu Glin Arg Luell Ala Ala Lieu. Glin Gly Arg Luell Glu Gly Luell Gly Ser 37 O 375

Ser Glu Ala Asp Glin Asp Gly Lieu Ala Ser Thir Wall Arg Ser Luell Gly 385 390 395 4 OO

Glu Thir Glin Luell Val Lieu. Tyr Gly Asp Wall Glu Glu Lell Arg Ser 4 OS 415

Wall Gly Glu Luell Pro Ser Thir Wall Glu Ser Luell Glin Wall Glin Glu 425 43 O

Glin Wall His Thir Lieu Lleu Ser Glin Asp Glin Ala Glin Ala Ala Arg Luell 435 44 O 445

Pro Pro Glin Asp Phe Lieu. Asp Arg Luell Ser Ser Lell Asp Asn Luell 450 45.5 460

Ala Ser Wall Ser Glin Wall Glu Ala Asp Luell Lys Met Lell Arg Thir Ala 465 470 475 48O

Wall Asp Ser Luell Val Ala Tyr Ser Wall Lys Ile Glu Thir Asn Glu Asn 485 490 495

Asn Luell Glu Ser Ala Lys Gly Lieu. Luell Asp Asp Lell Arg Asn Asp Luell SOO 505

Asp Arg Luell Phe Val Llys Val Glu Ile His Glu Lys Wall 515 52O 525

<210s, SEQ ID NO 13 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 13

Met Pro Ser Ala Lys Glin Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1. 5 15

Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Wall 2O 25 3O

Ala Lys Pro Pro Pro Ala Pro Glin Pro Pro Pro Pro Pro Ala 35 4 O 45

Pro Pro Glin Gln His Pro Glin His Pro Glin Asn Glin Ala His 55 6 O

Gly Gly Gly His Arg Gly Gly Gly Gly Gly Gly Ser Ser 65 70 7s 8O

Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ser Ser 85 90 95

Ser Ala Ser Cys Ser Arg Arg Lieu. Arg Ala Lell Asn Phe Luell Phe 11 O

Luell Ala Luell Wall Ala Ala Ala Phe Ser Gly Trp Wall His 115 12 O 125 US 9,075,045 B2 101 102 - Continued

His Wall Luell Glu Glu Wall Glin Glin Wall Arg Arg Ser His Glin Asp Phe 13 O 135 14 O

Ser Arg Glin Arg Glu Glu Lell Gly Glin Gly Luell Glin Gly Wall Glu Glin 145 150 155 160

Wall Glin Ser Lell Glin Ala Thir Phe Gly Thir Phe Glu Ser Ile Luell 1.65 17O 17s

Arg Ser Ser Glin His Glin Asp Luell Thir Glu Ala Wall Glin 18O 185 19 O

Gly Glu Ser Glu Wall Ser Arg Ile Ser Glu Wall Lell Glin Luell Glin 195

Asn Glu Ile Luell Lys Asp Lell Ser Asp Gly Ile His Wall Wall Asp 21 O 215 22O

Ala Arg Glu Arg Asp Phe Thir Ser Luell Glu ASn Thir Wall Glu Glu Arg 225 23 O 235 24 O

Lell Thir Glu Luell Thir Ser Ile Asn Asp ASn Ile Ala Ile Phe Thir 245 250 255

Glu Wall Glin Lys Arg Ser Glin Glu Ile ASn Asp Met Lys Ala Lys 26 O 265 27 O

Wall Ala Ser Luell Glu Glu Ser Glu Gly Asn Glin Asp Luell Ala 27s 28O 285

Lell Lys Glu Ala Wall Glu Ile Glin Thir Ser Ala Ser Arg Glu 29 O 295 3 OO

Trp Asp Met Glu Ala Lell Arg Ser Thir Luell Glin Thir Met Glu Ser Asp 3. OS 310 315

Ile Thr Glu Wall Arg Glu Lieu Wall Ser Lieu. Gln Glu Gln Gln 3.25 330 335

Ala Phe Glu Ala Ala Asp Thir Glu Arg Luell Ala Lell Glin Ala Luell 34 O 345 35. O

Thir Glu Lys Luell Lell Arg Ser Glu Glu Ser Wall Ser Arg Luell Pro Glu 355 360 365

Glu Ile Arg Arg Lell Glu Glu Glu Luell Arg Glin Lell Ser Asp Ser 37 O 375

His Gly Pro Glu Asp Gly Gly Phe Arg His Ser Glu Ala Phe Glu 385 390 395 4 OO

Ala Luell Glin Glin Lys Ser Glin Gly Luell Asp Ser Arg Lell Glin His Wall 4 OS 415

Glu Asp Gly Wall Lell Ser Met Glin Wall Ala Ser Ala Arg Glin Thir Glu 425 43 O

Ser Luell Glu Ser Lell Lell Ser Lys Ser Glin Glu His Glu Glin Arg Luell 435 44 O 445

Ala Ala Luell Glin Gly Arg Lell Glu Gly Luell Gly Ser Ser Glu Ala Asp 450 45.5 460

Glin Asp Gly Luell Ala Ser Thir Wall Arg Ser Luell Gly Glu Thir Glin Luell 465 470

Wall Luell Gly Asp Wall Glu Glu Luell Lys Arg Ser Wall Gly Glu Luell 485 490 495

Pro Ser Thir Wall Glu Ser Lell Glin Lys Wall Glin Glu Glin Wall His Thir SOO 505

Lell Luell Ser Glin Asp Glin Ala Glin Ala Ala Arg Lell Pro Pro Glin Asp 515 525

Phe Luell Asp Arg Lell Ser Ser Luell Asp Asn Luell Lys Ala Ser Wall Ser 53 O 535 54 O

Glin Wall Glu Ala Asp Lell Met Luell Arg Thir Ala Wall Asp Ser Luell US 9,075,045 B2 103 104 - Continued

5.45 550 555 560

Val Ala Tyr Ser Val Lys Ile Glu Thir Asn. Glu ASn Asn Lieu. Glu Ser 565 st O sts

Ala Lys Gly Lieu. Lieu. Asp Asp Lieu. Arg Asn Asp Lieu. Asp Arg Lieu. Phe 585 59 O

Val Llys Val Glu Lys Ile His Glu Lys Wall 595

<210s, SEQ ID NO 14 &211s LENGTH: 424 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 14

Met Pro Ser Ala Lys Glin Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1. 5 15

Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Wall 25 3O

Ala Lys Pro Pro Pro Ala Pro Glin Pro Pro Pro Pro Pro Ala 35 4 O 45

Pro Pro Glin Gln His Pro Glin His Pro Glin Asn Glin Ala His 55 6 O

Gly Gly Gly His Arg Gly Gly Gly Gly Gly Gly Ser Ser 65 70

Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ser Ser 85 90 95

Ser Ala Ser Cys Ser Arg Arg Lieu. Arg Ala Lell Asn Phe Luell Phe 11 O

Luell Ala Luell Wall Ala Ala Ala Phe Ser Gly Trp Wall His 115 12 O 125

His Wall Luell Glu Glu Wall Glin Glin Wall Arg Arg Ser His Glin Asp Phe 13 O 135 14 O

Ser Arg Glin Arg Glu Glu Lieu. Gly Glin Gly Luell Glin Gly Wall Glu Glin 145 150 155 160

Wall Glin Ser Lieu. Glin Ala Thr Phe Gly Thir Phe Glu Ser Ile Luell 1.65 17O 17s

Arg Ser Ser Glin His Lys Glin Asp Luell Thir Glu Ala Wall Glin 18O 185 19 O

Gly Glu Ser Glu Val Ser Arg Ile Ser Glu Wall Lell Glin Luell Glin 195 2OO

Asn Glu Ile Luell Lys Asp Lieu. Ser Asp Gly Ile His Wall Wall Asp 21 O 215 22O

Ala Arg Glu Arg Asp Phe Thir Ser Luell Glu ASn Thir Wall Glu Glu Arg 225 23 O 235 24 O

Lell Thir Glu Luell Thr Lys Ser Ile Asn Asp ASn Ile Ala Ile Phe Thir 245 250 255

Glu Ser Luell Luell Ser Lys Ser Glin Glu His Glu Glin Arg Luell Ala Ala 26 O 265 27 O

Lell Glin Gly Arg Lieu. Glu Gly Lieu. Gly Ser Ser Glu Ala Asp Glin Asp 28O 285

Gly Luell Ala Ser Thr Val Arg Ser Luell Gly Glu Thir Glin Luell Wall Luell 29 O 295 3 OO

Tyr Gly Asp Wall Glu Glu Lieu Lys Arg Ser Wall Gly Glu Luell Pro Ser 3. OS 310 315 32O US 9,075,045 B2 105 106 - Continued

Thir Wall Glu Ser Lieu. Glin Llys Val Glin Glu Glin Wall His Thir Luell Luell 3.25 330 335

Ser Glin Asp Glin Ala Glin Ala Ala Arg Luell Pro Pro Glin Asp Phe Luell 34 O 345 35. O

Asp Arg Luell Ser Ser Lieu. Asp Asn Luell Lys Ala Ser Wall Ser Glin Wall 355 360 365

Glu Ala Asp Luell Llys Met Lieu. Arg Thir Ala Wall Asp Ser Luell Wall Ala 37 O 375

Tyr Ser Wall Ile Glu. Thir Asn Glu Asn ASn Lell Glu Ser Ala Lys 385 390 395 4 OO

Gly Luell Luell Asp Asp Lieu. Arg Asn Asp Luell Asp Arg Lell Phe Wall Lys 4 OS 41O 415

Wall Glu Ile His Glu Lys Val 42O

<210s, SEQ ID NO 15 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15

Met Pro Ser Ala Lys Glin Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1. 5 15

Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Wall 25 3O

Ala Lys Pro Pro Pro Ala Pro Glin Pro Pro Pro Pro Pro Ala 35 4 O 45

Pro Pro Glin Gln His Pro Glin His Pro Arg Asn Glin Ala His 55 6 O

Gly Gly Gly His Arg Gly Gly Gly Gly Gly Gly Ser Ser 65 70

Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ser Ser 85 90 95

Ser Ala Ser Cys Ser Arg Arg Lieu. Arg Ala Lell Asn Phe Luell Phe 11 O

Luell Ala Luell Wall Ala Ala Ala Phe Ser Gly Trp Wall His 115 12 O 125

His Wall Luell Glu Glu Wall Glin Glin Wall Arg Arg Ser His Glin Asp Phe 13 O 135 14 O

Ser Arg Glin Arg Glu Glu Lieu. Gly Glin Gly Luell Glin Gly Wall Glu Glin 145 150 155 160

Wall Glin Ser Lieu. Glin Ala Thr Phe Gly Thir Phe Glu Ser Ile Luell 1.65 17O 17s

Arg Ser Ser Glin His Lys Glin Asp Luell Thir Glu Ala Wall Glin 18O 185 19 O

Gly Glu Ser Glu Val Ser Arg Ile Ser Glu Wall Lell Glin Luell Glin 195 2OO

Asn Glu Ile Luell Lys Asp Lieu. Ser Asp Gly Ile His Wall Wall Asp 21 O 215 22O

Ala Arg Glu Arg Asp Phe Thir Ser Luell Glu ASn Thir Wall Glu Glu Arg 225 23 O 235 24 O

Lell Thir Glu Luell Thr Lys Ser Ile Asn Asp ASn Ile Ala Ile Phe Thir 245 250 255

Glu Wall Glin Lys Arg Ser Gln Lys Glu Ile ASn Asp Met Lys Ala Lys 26 O 265 27 O US 9,075,045 B2 107 108 - Continued

Wall Ala Ser Luell Glu Glu Ser Glu Gly Asn Glin Asp Lieu Lys Ala 27s 28O 285

Lell Lys Glu Ala Wall Glu Ile Glin Thir Ser Ala Lys Ser Arg Glu 29 O 295 3 OO

Trp Asp Met Glu Ala Lell Arg Ser Thir Luell Glin Thir Met Glu Ser Asp 3. OS 310 315

Ile Thir Glu Wall Arg Glu Luell Wall Ser Luell Glin Glu Glin Glin 3.25 330 335

Ala Phe Glu Ala Ala Asp Thir Glu Arg Luell Ala Lell Glin Ala Luell 34 O 345 35. O

Thir Glu Lys Luell Lell Arg Ser Glu Glu Ser Wall Ser Arg Luel Pro Glu 355 360 365

Glu Ile Arg Arg Lell Glu Glu Glu Luell Arg Glin Lell Ser Asp Ser 37 O 375

His Gly Pro Glu Asp Gly Gly Phe Arg His Ser Glu Ala Phe Glu 385 390 395 4 OO

Ala Luell Glin Glin Lys Ser Glin Gly Luell Asp Ser Arg Lell Gln His Wall 4 OS 415

Glu Asp Gly Wall Lell Ser Met Glin Wall Ala Ser Ala Arg Gn. Thir Glu 425 43 O

Ser Luell Glu Ser Lell Lell Ser Lys Ser Glin Glu His Glu Glin Arg Luell 435 44 O 445

Ala Ala Luell Glin Gly Arg Lell Glu Gly Luell Gly Ser Ser Glu Ala Asp 450 45.5 460

Glin Asp Gly Luell Ala Ser Thir Wall Arg Ser Luell Gly Glu Thr Gin Luell 465 470

Wall Luell Gly Asp Wall Glu Glu Luell Lys Arg Ser Wall Gly Glu Luell 485 490 495

Pro Ser Thir Wall Glu Ser Lell Glin Lys Wall Glin Glu Glin Wall His Thir SOO 505 51O

Lell Luell Ser Glin Asp Glin Ala Glin Ala Ala Arg Lell Pro Pro Glin Asp 515 525

Phe Luell Asp Arg Lell Ser Ser Luell Asp Asn Luell Lys Ala Ser Wall Ser 53 O 535 54 O

Glin Wall Glu Ala Asp Lell Met Luell Arg Thir Ala Wall Asp Ser Luell 5.45 550 555 560

Wall Ala Ser Wall Ile Glu Thir Asn Glu Asn Asn Lieu. Glu Ser 565 st O sts

Ala Gly Luell Lell Asp Asp Luell Arg Asn Asp Lell Asp Arg Lieu. Phe 585 59 O

Wall Wall Glu Lys Ile His Glu Lys Wall 595 6OO

SEQ ID NO 16 LENGTH: TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 16

Met Pro Ser Ala Lys Glin Arg Gly Ser Lys Gly Gly His Gly Ala Ala 1. 5 15

Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Wall 25 3O

Ala Lys Llys Pro Pro Pro Ala Pro Glin Gln Pro Pro Pro Pro Pro Ala US 9,075,045 B2 109 110 - Continued

35 4 O 45

Pro His Pro Glin Glin His Pro Glin Glin His Pro Glin Asn Glin Ala His SO 55 6 O

Gly Gly Gly His Arg Gly Gly Gly Gly Gly Gly Gly Ser Ser 65 70

Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser 85 90 95

Ser Ala Ser Cys Ser Arg Arg Luell Gly Arg Ala Lell Asn Phe Luell Phe 105 11 O

Luell Ala Luell Wall Ala Ala Ala Ala Phe Ser Gly Trp Wall His 115 12 O 125

His Wall Luell Glu Glu Wall Glin Glin Wall Arg Arg Ser His Glin Asp Phe 13 O 135 14 O

Ser Arg Glin Arg Glu Glu Lell Gly Glin Gly Luell Glin Gly Wall Glu Glin 145 150 155 160

Wall Glin Ser Lell Glin Ala Thir Phe Gly Thir Phe Glu Ser Ile Luell 1.65 17O 17s

Arg Ser Ser Glin His Glin Asp Luell Thir Glu Ala Wall Glin 18O 185 19 O

Gly Glu Ser Glu Wall Ser Arg Ile Ser Glu Wall Lell Glin Luell Glin 195

Asn Glu Ile Luell Asp Lell Ser Asp Gly Ile His Wall Wall Asp 21 O 215 22O

Ala Arg Glu Arg Asp Phe Thir Ser Luell Glu ASn Thir Wall Glu Glu Arg 225 23 O 235 24 O

Lell Thir Glu Luell Thir Ser Ile Asn Asp ASn Ile Ala Ile Phe Thir 245 250 255

Glu Wall Glin Lys Arg Ser Glin Glu Ile ASn Asp Met Lys Ala 26 O 265 27 O

Wall Ala Ser Luell Glu Glu Ser Glu Gly Asn Lys Glin Asp Luell Ala 27s 28O 285

Lell Lys Glu Ala Wall Glu Ile Glin Thir Ser Ala Ser Arg Glu 29 O 295 3 OO

Trp Asp Met Glu Ala Lell Arg Ser Thir Luell Glin Thir Met Glu Ser Asp 3. OS 310 315

Ile Thir Glu Wall Arg Glu Luell Wall Ser Luell Glin Glu Glin Glin 3.25 330 335

Ala Phe Glu Ala Ala Asp Thir Glu Arg Luell Ala Lell Glin Ala Luell 34 O 345 35. O

Thir Glu Lys Luell Lell Arg Ser Glu Glu Ser Wall Ser Arg Luell Pro Glu 355 360 365

Glu Ile Arg Arg Lell Glu Glu Glu Luell Arg Glin Lell Ser Asp Ser 37 O 375 38O

His Gly Pro Glu Asp Gly Gly Phe Arg His Ser Glu Ala Phe Glu 385 390 395 4 OO

Ala Luell Glin Glin Lys Ser Glin Gly Luell Asp Ser Arg Lell Glin His Wall 4 OS 41O 415

Glu Asp Gly Wall Lell Ser Met Glin Wall Ala Ser Ala Arg Glin Thir Glu 42O 425 43 O

Ser Luell Glu Ser Lell Lell Ser Lys Ser Glin Glu His Glu Glin Arg Luell 435 44 O 445

Ala Ala Luell Glin Gly Arg Lell Glu Gly Luell Gly Ser Ser Glu Ala Asp 450 45.5 460

US 9,075,045 B2 123 124 - Continued cct caggact tcc toggacag actitt cittct c taga caacc togaaag.cctic agt cago caa SOO gtggaggcgg acttgaaaat gct caggact gctgtggaca gtttggttgc atact cqgt C 560 aaaatagaaa C caacgagaa caatctggaa t cagccalagg gtt tact aga tigacctgagg 62O aatgat Ctgg at aggttgtt ttgaaagtg gagaagattic acgaaaaggt Ctaaatgaat 68O tgcgtgtgca gggcgcggat ttaaagtc.ca atttct catg accaaaaaat gtgtggittitt 74 O titcc catgtg toccct acco cocaatttct tdtcc cct ct taaagagcag ttgtcaccac 8OO ctgaacacca aggcattgta ttitt catgcc cagittaactt atttacaata tittaagttct 86 O Ctgcttctgc atttggttgg ttt Cotgaag cqcagcc cct gtgaataa.ca ggtggcttitt 92 O

Catggatgtc. tctagt caga gaaaaatgat aaaggcttaa attgaggatt alacagaa.gca 98 O gattaacctic agaaatcc td totggctggc agatttcaag taaaaaaaaa aaaaaaaaa 2O39

What is claimed is: by a process selected from the group consisting of fluores 1. A method for diagnosis of interstitial cystitis in a patient cence resonance electron transfer (FRET), lanthanide reso by detecting the presence of APF in a biological sample nance electron transfer (LRET), fluorescence cross-correla comprising: tion spectroscopy, fluorescence quenching, fluorescence polarization, flow cytometry, Scintillation proximity, lumi (a) providing a diagnostic system, wherein said diagnostic 25 system comprises one or more detection reagents nescence resonance energy transfer, direct quenching, Selected from the group consisting of: ground-state complex formation, chemiluminescence energy a first detection reagent which is a fragment of CKAP4 transfer, bioluminescence resonance energy transfer, excimer which comprises a polyhistidine tag at the end of said formation, colorimetric Substrates detection, phosphores cence, electro-chemical changes, and redox potential fragment of CKAP4, and wherein the first detection 30 reagent specifically binds antiproliferative factor (APF), changes. 3. The method of claim 1, wherein the visual indication is further wherein the first detection reagent is detectably detected following APF binding due to a change in the con labeled, wherein the binding of APF to the detection formation of the CKAP4 receptor, or a change in correspond reagent generates a signal that provides detection of a ing protein-protein interactions. predetermined threshold level of APF in the sample in 35 the form of a visual indication; 4. The method of claim 1, wherein the visual indication is a second detection reagent which is a fragment of CKAP4 provided by a process selected from the group consisting of FRET, BRET, LRET and LET may be detected either which comprises a polyhistidine tag at the end of said intramolecularly, where the donor and acceptor fluorophores fragment of CKAP4, and wherein the second detection are part of the same sensor molecule or intermolecularly reagent specifically binds antiproliferative factor (APF), 40 further wherein the second detection reagent is detect where the donor and acceptor are physically associated with ably labeled, wherein the binding of APF to the detection two separate molecules. reagent generates a signal that provides detection of a 5. The method according to claim 1, wherein said diagnos tic system is a single device which comprises both the first predetermined threshold level of APF in the sample in and second diagnostic reagents. the form of a visual indication; and 45 both said first and said second detection reagents; 6. The method according to claim 1, wherein said diagnos (b) providing a biological sample from a patient; tic system comprises more than one device. (c) contacting the first detection reagent with the biological 7. The method according to claim 1, wherein the diagnostic sample to provide a patient test sample; system is a device which comprises a housing having a cham ber consisting of one or more test wells disposed inside the (d) providing a control sample, wherein the control sample 50 is from a subject without interstitial cystitis; housing and having an inlet above each well to allow the (e) contacting the second detection reagent with the control introduction of the biological sample, optionally wherein sample to provide a normal control test sample: each test well contains a detection reagent. 8. The method according to claim 7, wherein the diagnostic (f) visualizing the diagnostic system to ascertain the signal system housing may be at least partially transparent, or has generated by APF binding to the first detection reagent 55 in the patient test sample: windows provided therein, for observation of an indicator (g) visualizing the diagnostic system to ascertain the signal region that undergoes a color or fluorescence change. 9. The method according to claim 1, wherein the diagnostic generated by APF binding to the second detection system is a device which is selected from the group consisting reagent in the normal control test sample, of a dipstick, a lateral flow device, and migration-type device. wherein a visually detectable greater signal of APF binding 60 in the patient test sample relative to APF binding in the 10. The method according to claim 9, wherein the diagnos normal control test sample is a positive screen for inter tic system is a lateral flow device comprising a housing hav Stitial cystitis in the patient. ing an inlet for the sample and side walls defining a fluid 2. The method of claim 1, wherein the binding of APF to the lateral flow path extending from the inlet. first or second detection reagent provides a visual indication k k k k k