METHODS AND COMPOSITIONS FOR DIAGNOSING HEPATOCELLULAR CARCINOMA

Abstract

of hepatocellular carcinoma (HCC) are set forth. Improved assay methods and scanning methods are included that employ non-cell-associated and cell-associated HCC related proteins. Such methods are based upon the discovery of genes that were up-regulated in diseased versus normal tissue as well as in HCC tissue when compared to the tissue of patients with other ailments.

Description

[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 60/393,982 filed on Jul. 3, 2002, hereby incorporated in its entirety by reference.

BACKGROUND

[0003]The field of the invention is the diagnosis of hepatocellular carcinoma (HCC).

[0004]Hepatocellular carcinoma (HCC) is the most prevalent form of liver cancer worldwide. Incidence of the disease varies geographically from between 1 in 5,000 in Asia to 1 in 20,000 in western nations (Wildi et al., 2002). Patients with chronic liver disease are at increased risk for development of hepatocellular carcinoma. This is particularly true for individuals with liver cirrhosis who should be closely monitored for development of this disease.

[0005]Currently, it is difficult to diagnose HCC. Methods employed generally rely on imaging techniques such as MRI, CT, and ultrasound and are of little use in detecting the disease in its earliest stages. As with most cancers, early detection of HCC would leave physicians with more treatment options and patients with a better prognosis (Befeler and Bisceglie, 2002).

[0006]Better imaging reagents would enhance the sensitivity and broaden the applicability of currently used scanning methodologies. Proteins expressed specifically or preferentially on the surface of HCC cells could be targeted by an antibody or other targeting reagent that is conjugated to an imaging agent. Such conjugates would aid in diagnosis of the disease at an early stage.

[0007]The literature describes a few serodiagnostic markers indicative of HCC, including alpha-fetoprotein (AFP), Lens culinaris agglutinin-reactive fraction (AFP-L3), and des-gamma-carboxy prothrombin or PIVKA-II (Shimizu et al., 2002; Ikoma et al., 2002; Fujiyama et al., 1986; Naraki et al., 2002). Unfortunately, at best, elevated levels of these serum proteins are detected in only about 50% of HCC patients. A significant increase in the sensitivity of HCC diagnosis can be achieved by combining tests for AFP, AFP-L3 and PIVKA-II. However, even when all three tests are combined, the sensitivity is only about 87% (Fujiyama et al., 2002).

[0008]Identification of new serodiagnostic markers specific to HCC and present in a large percentage of HCC patients would greatly improve the diagnosis of this disease and be more cost effective than commonly used scanning methodologies and/or the combined use of all currently available serodiagnostic assays.

[0009]These and other limitations and problems of the past are solved by the present invention.

BRIEF SUMMARY OF THE INVENTION

[0010]The present invention relates to the detection of hepatocellular carcinoma (HCC) by assaying patient samples such as tissue, plasma, serum, etc. for the presence and level of specific HCC related proteins. Some of these proteins will be cell associated, while others will not be cell associated. A finding of elevated levels of one or more of these proteins in a patient sample indicates that the patient has hepatocellular carcinoma. HCC diagnosis based on quantification of the HCC related protein(s) will be dependent upon research that will define a variety of parameters. These parameters will include: (a) a determination of the relative levels of the HCC related proteins in diseased versus normal patient samples (as an example of a control level, but not limited to), and (b) the specificity, sensitivity and reproducibility of the assay or assays employed.

[0011]The present invention also relates to identification of tumor markers that may be targeted by specific reagents to enhance early diagnosis of HCC by traditional scanning methodologies. Proteins expressed specifically on the surface of HCC cells could be targeted by an antibody or other targeting reagent (e.g. soluble receptor or ligand) that binds specifically to the cell-associated HCC protein. The targeting moiety is conjugated to an imaging agent to enable visualization of the construct.

[0012]The proteins that are useful in accordance with the present invention are: phospholipase A2 (Group XIII) (SEQ ID Nos. 1-2); phospholipase A2 (group VII) (SEQ ID No. 12); anti-thrombin III (SEQ ID No. 3); apolipoprotein B (SEQ ID No. 4); group C specific vitamin D binding protein (SEQ ID Nos. 5-6); gamma-glutymyl hydrolase (SEQ ID No. 7); nicastrin (SEQ ID No. 8); pregnancy associated plasma protein A, plasma glutamate carboxypeptidase (SEQ ID No. 11); secretory carrier membrane protein-3 (SEQ ID Nos. 9-10); and other hypothetical proteins described herein. Not all of the proteins that are useful within the methods of the present invention are found exclusively in HCC patients. Some proteins will be found in both patients with and without HCC. In these cases, HCC affected individuals will be distinguished from non diseased individuals by a significant elevation in the amount of one or more of the proteins described in the current invention.

[0013]The invention will best be understood by reference to the following detailed description of the preferred embodiment. The discussion below is descriptive, illustrative and exemplary and is not to be taken as limiting the scope defined by any appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0014]Expression microarray analysis of tumor samples from Hepatitis C(HCV) infected patients with hepatocellular carcinoma (HCC) led to the identification of genes that were specifically up-regulated in hepatocellular carcinoma tumor tissue when compared to HCV infected cirrhotic non-tumor tissue, and normal liver.

[0015]Liver and HCC samples were obtained during surgical procedures with prior informed consent from all persons involved. HCC samples included 21 from HCV infected patients and 1 from a patient infected with Hepatitis B. In addition, 4 samples of normal, non-diseased liver and 8 samples of HCV infected, cirrhotic liver with no evidence of HCC were used for analysis.

[0016]Total RNA was isolated as described in Geiss et al. (2001). RNA amplification was performed using a T7 RNA polymerase protocol (Eberwine, 1996) with the AmpliScribe Transcription kit (Epicentre Technologies, Madison, Wis.) as described by the manufacturer. The quality of amplified RNA samples was evaluated using capillary electrophoresis in an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif.).

[0017]cDNA microarrays were constructed by the University of Washington's Center for Expression Array Technology using PCR products generated by amplification of sequence verified I.M.A.G.E. consortium clones obtained from Research Genetics (St. Louis, Mo.) (Lennon et al. 1996). Microarrays were constructed as previously described (Geiss et al. 2001). A human high density set consisted of two arrays, each of which represented 7,296 human clones in duplicate with a number of additional control sequences, for a total of 14,976 clones (approximately 13,597 unique I.M.A.G.E. cDNA clones). Each single experiment involved interrogation of two slides for which the dye labels had been reversed (fluor reversal methodology as described in Geiss et al., 2000; Geiss et al., 2001). A total of at least four separate hybridization measurements were taken per gene per experiment.

[0018]Protocols for probe synthesis, microarray hybridization, and wash conditions are as previously described (Geiss et al. 2001). Microarrays were scanned and the images were quantified using a custom spot-finding program, Spot-On Image (Geiss et al, 2000 and Geiss et al., 2001), that calculated the standard deviations and the mean ratios between the expression levels of each gene in the analyzed pair of samples. Raw data and sample information were entered into a custom designed database, Expression Array Manager, and evaluated using Rosetta Biosoftware's Resolver® Version 3.0 (Rosetta Biosoftware, Kirkland, Wash.), a software package for the storage and analysis of microarray expression data. This package implements common statistical procedures (clustering, trend analysis, similarity searches based on a BLAST-related algorithm, etc.) together with a sophisticated error model to compensate for biological and experimental variation.

[0019]The expression microarray data was processed by two different methods. The first involved examining only HCV-infected HCC patient samples and sorting for genes that were significantly (p<0.01) up-regulated more than two-fold in tumor versus non-tumor liver samples from the same patient. Genes that met these criteria in ten or more patients were then analyzed relative to samples from HCV infected patients with liver cirrhosis but no tumors and also relative to samples of normal healthy liver. If the gene was unchanged or down-regulated in these control samples, its potential for use as a diagnostic target was further evaluated using information available in the National Center for Biotechnology Information databases (Unigene, OMIM, LocusLink, and HomoloGene) and currently published literature regarding the location and function of its protein product. The protein products of the genes that meet the above criteria and are (a) secreted or likely to be present on the plasma membrane and are (b) noted to be preferentially or specifically expressed in liver, are likely to be diagnostic indicators of HCC. The following are an example of some of these proteins while their corresponding amino acid sequences and variants thereof are included in the sequence listing accompanying this application:

PGLA2G13 (phospholipase A2 Group XIII; IMAGE EST: 297107; GenBank AF349540; Unigene: 333175; mRNA: NM 032562; protein: NP 115951; (SEQ ID Nos. 1-2)); SERPINC1 (serine or cysteine proteinase inhibitor; anti thrombin III; IMAGE EST: 85643; GenBank X68793; Unigene: Hs.75599; mRNA: 000488; protein: NP 000479; (SEQ ID No. 3));APOB (apolipoprotein B; IMAGE EST: 206632; GenBank X04506; Unigene: Hs.585; mRNA: NM 000384; protein: NP 000375; (SEQ ID No. 4));GC (group C specific vitamin D binding protein; IMAGE EST: 195340; GenBank M12654; Unigene: Hs.198246; mRNA: NM 000583; protein: NP 000574; (SEQ ID Nos. 5-6));GGH (gamma-glutymyl hydrolase; conjugase; folylpolygammaglutamyl hydrolase; IMAGE EST: 809588; GenBank U55206; Unigene: Hs.78619; mRNA: NM 003878; protein: NP 003869; (SEQ ID No. 7)); andNCSTN (nicastrin; IMAGE EST: 199645; GenBank R96527; Unigene: Hs.4788; (SEQ ID No. 8)).

[0020]The function of a number of genes that were up-regulated in the HCC samples but not in control samples is unknown. Included herein are the protein products of these genes and their use as diagnostic markers for HCC. These gene products are as follows:

Protein coded by the gene specified as: IMAGE EST: 241475; GenBank H90421;

Unigene: Hs.41407;

[0021]Protein coded by the gene specified as: IMAGE EST: 293094; GenBank N91620;

Unigene: Hs.12160;

[0022]Protein coded by the gene specified v: IMAGE EST: 430221; GenBank AA010360;

Unigene: Hs.60380;

[0023]Protein coded by the gene specified as: IMAGE EST: 52990; GenBank R15441;

Unigene: Hs.4774;

[0024]Protein coded by gene specified as: IMAGE EST: 153779; GenBank R48248; Unigene: Hs.183171; mRNA: NM 024838; protein: NP 079114 hypothetical protein FLJ22002; (SEQ ID No. 13).

[0025]The second method of processing the microarray data yielded similar results. Error probabilities were used to filter the initial 13,597 member gene set to a set of 2302 genes that demonstrated differential regulation of two-fold or greater with 95% confidence (p<=0.05) in at least 4 out of 20 experiments involving the comparison of HCC tumor versus matched non-tumor tissues. A keyword search was then applied to this group to identify genes encoding putative secreted and/or plasma membrane proteins. The resultant small gene subset was manually filtered to exclude those genes that were down-regulated in most tumors. Finally, a set of 11 genes was selected and used for two dimensional clustering analyses of all 4 experiments. Four out of 11 genes showed a pronounced up-regulation of gene expression in about 60 to 70% of all tumor versus non-tumor liver experiments. Also, all four genes were significantly up-regulated in experiments involving pooled tumor versus normal liver samples. The four gene products are listed below and include several of the proteins noted above. Corresponding amino acid sequences and variants thereof are listed in the sequence listing accompanying this patent.

SCAMP3 (secretory carrier membrane protein-3; IMAGE EST: 156045; GenBank R72518, Unigene: Hs.200600; mRNA: NM 005698; protein: NP 005689; (SEQ ID Nos. 9-10));PGCP (plasma glutamate carboxypeptidase; IMAGE EST: 796263; Unigene: Hs.197335; (SEQ ID No. 11)) Gingras et al. 1999;PGLA2G13 (phospholipase A2 Group XIII; IMAGE EST: 297107; GenBank AF349540;Unigene: 333175; mRNA: NM 032562; protein: NP 115951; (SEQ ID Nos. 1-2)); andPLA2G7 (phospholipase A2 group VII; IMAGE EST: 238821; GenBank H65029;Unigene: Hs.93304; mRNA: NM 005084; protein: NP 005075; (SEQ ID No. 12)).

[0026]Several of the proteins that were identified by either method will find use for the diagnosis of HCC. An elevated level of one or more of these proteins in a patient sample is indicative of disease. Diagnostic proteins are expressed in either a cell associated or non-cell associated way. The method of diagnosis will depend on whether the diagnostic or predictive protein is cell associated or non-cell associated.

[0027]The non-cell associated proteins include PGCP (SEQ ID No. 11), PGLA2G13 (SEQ ID Nos. 1-2), PLA2G7 (SEQ ID No. 12), SERPINC1 (SEQ ID No. 3), APOB (SEQ ID No. 4), GC (SEQ ID Nos. 5-6), and GGH (SEQ ID No. 7). The diagnosis of HCC may result from quantification of these proteins individually or in combination in patient samples such as blood, plasma, serum, urine, etc.

[0028]The presence and quantity of non-cell associated proteins within a patient sample will be measured by state of the art techniques which include, but are not limited to, ELISA, sandwich ELISA, radiolabeled immunoassay (RIA) or other competitive binding assay that is based on the use of specific antibodies. Alternatively, activity assays for quantification of those non-cell associated proteins that are enzymes (PGCP (SEQ ID No. 11); PLA2G7 (SEQ ID No. 12); PLA2G13 (SEQ ID Nos. 1-2); SERPINC1 (SEQ ID No. 3); and GGH (SEQ ID No. 7)) may also be employed.

[0029]In addition or in the alternative, HCC may be diagnosed by imaging or scanning methodologies employing targeting agent-imaging agent conjugates. Preferred proteins for this aspect of the present invention are the cell associated proteins, SCAMP3 (SEQ ID Nos. 9-10) and NCSTN (SEQ ID No. 8), and will find use as imaging targets when used in combination with labeling and scanning technologies.

[0030]The targeting agents useful in the practice of the present invention include, but are not limited to, antibodies or soluble receptors or ligands or other agents that specifically bind proteins expressed by HCC cells. When conjugated to imaging agents, these targeting agents enable visualization of tumor cells.

[0031]The imaging agents useful in the practice of the present invention include, but are not limited to, radioisotopes, electron dense dyes and/or a variety of other reagents visible to scanning technologies that have been well described in the literature (see for example: Vera et al. 1995; Shen et al. 1996; Matsumura et al. 1994; Reimer et al. 1994; Koral et al. 1994; Winzelberg et al. 1992; Perkins et al. 1993).

[0032]The targeting molecule-imaging agent-conjugate will be administered to the patient intravenously prior to employment of the imaging application thereby enabling and/or enhancing tumor visualization. The molecular imaging agent-conjugate may bind to the cell associated HCC related protein or may be subject to receptor mediated uptake where the receptor is the cell associated HCC related protein.

[0033]Other methods of the present invention involve the use of liver tissue samples. For these aspects of the present invention, the patient sample may be obtained by biopsy or other technique known in the art.

[0034]An embodiment of the present invention useful in the analysis of tissue samples includes employing immunocytochemistry or immunohistochemistry techniques using a cell-associated HCC related protein specific antibody conjugated to imaging agents.

[0035]In addition, tissue samples may be evaluated by assaying for transcription of one or more of the cell-associated or non-cell associated HCC related proteins by RT-PCR or nucleic acid hybridization methods.

[0036]The diagnosis of HCC may result from quantification of these proteins individually or in combination using any of the methods noted above.

[0037]Of direct relevance herein are the development of polyclonal antibodies which bind to recombinant human PLA2G13 (SEQ ID Nos. 1-2) and the use of said antibodies in quantification or visualization of PLA2G13 (SEQ ID Nos. 1-2). The generation of polyclonal antisera by immunization of rabbits and the use of Western Blot analysis, as outlined below, will be familiar to one skilled in the art.

[0038]Polyclonal antibodies were generated by immunizing rabbits with either the recombinant human PLA2G13 (variant 1; SEQ ID No. 1) or with synthetic peptides (SEQ ID Nos. 14-16) representing portions of human PLA2G13 (SEQ ID No. 1) coupled to a carrier protein. The sequence of each of these peptides is indicated below with an additional cysteine residue added to the 5'-terminus of peptide #1 as a means of conjugation to the carrier protein.

TABLE-US-00001 (SEQ ID No. 14) Peptide #1: 5' CSDTSPDTEESYSD 3' (SEQ ID No. 15) Peptide #2: 5' CSDLKRSLGFVSKVE 3' (SEQ ID No. 16) Peptide #3: 5' CAEEEKEEL 3'

[0039]Antisera from rabbits immunized with recombinant human PLA2G13 (SEQ ID No. 1) or with carrier protein conjugates of peptides #1 or #3 contained antibodies that bound recombinant human PLA2G13 (SEQ ID No. 1). This was verified by a Western Blot Assay.

[0040]The recombinant human PLA2G13 (SEQ ID No. 1) used in Western Blot Assay was expressed in, and purified from E. coli using known molecular biological and biochemical methods as outlined in Koduri et al. (2002) for a similar protein. Additionally, the recombinant human PLA2G13 (SEQ ID No. 1) was refolded as and characterized as described for a similar protein by Valentin et al., 1999, indicating that it is in its native conformation. Polyclonal antibodies that bind the recombinant human PLA2G13 (SEQ ID No. 1) in a native conformation will likely bind endogenous or native PLA2G13 (SEQ ID No. 1-2) in humans or human derived material. The generation of polyclonal antibodies that bind PLA2G13 (SEQ ID No. 1) enables the development of antibody based assays to detect endogenous PLA2G13 (SEQ ID Nos. 1-2) in patients or detect and quantify PLA2G13 (SEQ ID Nos. 1-2) in patient derived material. Additionally, the anti-PLA2G13 (SEQ ID No. 1) antibodies can serve as the targeting portion of imaging conjugate(s).

[0041]The discussion above is descriptive, illustrative and exemplary and is not to be taken as limiting the scope defined by any appended claims.

REFERENCES

[0042]1) Fujiyama, S. et al. (2002) Oncology 62 (supp11): 57-63 [0043]2) Befeler, A. S. and Bisceglie, A. M. (2002) Gastroenterol. 122: 1609-1619 [0044]3) Eberwine, J. (1996) Biotechniques 20: 584-591 [0045]4) Fujiyama et al. (1986) Hepatogastroenterol. 33: 201-205 [0046]5) Geiss, G. K. et al. (2000) Virol. 266: 8-16 [0047]6) Geiss, G. K. et al. (2001) J. Virol. 75: 4321-4331 [0048]7) Gingras, R. et al. (1999) J. Biol. Chem. 274: 11742-11750 [0049]8) Ikoma, J. et al. (2002) Hepatogastroenterol. 49: 235-238 [0050]9) Koduri, R. S. et al. (2002) J. Biol. Chem. 277: 5849-5857 [0051]10) Koral, K. F. (1994) J. Nucl. Med. 35: 1714-1720 [0052]11) Lennon, G. et al. (1996) Genomics 33(1): 151-15 [0053]12) Matsumura, A. et al. (1994) Acta Neurochir. Suppl (WEIN) 60: 356-358 [0054]13) Naraki, T. et al. (2002) BBA 1586: 287-298 [0055]14) Orci, L. et al. (1989) Science 245: 295-297 [0056]15) Perkins, A. C. (1993) Nucl. Med. Commun. 14: 578-586 [0057]16) Reimer, P. et al. (1994) Radiology 193: 527-531 [0058]17) Shen, T. T. et al. (1996) Bioconjug. Chem. 7: 311-316 [0059]18) Shimizu, A. et al. (2002) International J. Mol. Med. 9: 245-250 [0060]19) Valentin, E. et al. (1999) J. Biol. Chem. 274: 19152-19160 [0061]20) Vera, D. R. et al. (1995) Acad. Radiol. 2: 497-506 [0062]21) Winzelberg, G. G. (1992) 69: 1656-1663 [0063]22) Wildi, S. et al. (2002) Swiss Surg. 8: 61-66

Sequence CWU 1

161195PRTHomo sapiens 1Met Lys Leu Ala Ser Gly Phe Leu Val Leu Trp Leu Ser Leu Gly Gly1 5 10 15Gly Leu Ala Gln Ser Asp Thr Ser Pro Asp Thr Glu Glu Ser Tyr Ser 20 25 30Asp Trp Gly Leu Arg His Leu Arg Gly Ser Phe Glu Ser Val Asn Ser 35 40 45Tyr Phe Asp Ser Phe Leu Glu Leu Leu Gly Gly Lys Asn Gly Val Cys 50 55 60Gln Tyr Arg Cys Arg Tyr Gly Lys Ala Pro Met Pro Arg Pro Gly Tyr65 70 75 80Lys Pro Gln Glu Pro Asn Gly Cys Gly Ser Tyr Phe Leu Gly Leu Lys 85 90 95Val Pro Glu Ser Met Asp Leu Gly Ile Pro Ala Met Thr Lys Cys Cys 100 105 110Asn Gln Leu Asp Val Cys Tyr Asp Thr Cys Gly Ala Asn Lys Tyr Arg 115 120 125Cys Asp Ala Lys Phe Arg Trp Cys Leu His Ser Ile Cys Ser Asp Leu 130 135 140Lys Arg Ser Leu Gly Phe Val Ser Lys Val Glu Ala Ala Cys Asp Ser145 150 155 160Leu Val Asp Thr Val Phe Asn Thr Val Trp Thr Leu Gly Cys Arg Pro 165 170 175Phe Met Asn Ser Gln Arg Ala Ala Cys Ile Cys Ala Glu Glu Glu Lys 180 185 190Glu Glu Leu 1952194PRTHomo sapiens 2Met Lys Leu Ala Ser Gly Phe Leu Val Leu Trp Leu Ser Leu Gly Gly1 5 10 15Gly Leu Ala Gln Ser Asp Thr Ser Pro Asp Thr Glu Glu Ser Tyr Ser 20 25 30Asp Trp Gly Leu Arg His Leu Arg Gly Ser Phe Glu Ser Val Asn Ser 35 40 45Tyr Phe Asp Ser Phe Leu Glu Leu Leu Gly Gly Lys Asn Gly Val Cys 50 55 60Gln Tyr Arg Cys Arg Tyr Gly Lys Ala Pro Met Pro Arg Pro Gly Tyr65 70 75 80Lys Pro Gln Glu Pro Asn Gly Cys Gly Ser Tyr Phe Leu Gly Leu Lys 85 90 95Val Pro Glu Ser Met Asp Leu Gly Ile Pro Ala Met Thr Lys Cys Cys 100 105 110Asn Gln Leu Asp Val Cys Tyr Asp Thr Cys Gly Ala Asn Lys Tyr Arg 115 120 125Cys Asp Ala Lys Phe Arg Trp Cys Leu His Ser Ile Cys Ser Asp Leu 130 135 140Lys Arg Ser Leu Gly Phe Val Ser Lys Val Glu Ala Cys Asp Ser Leu145 150 155 160Val Asp Thr Val Phe Asn Thr Val Trp Thr Leu Gly Cys Arg Pro Phe 165 170 175Met Asn Ser Gln Arg Ala Ala Cys Ile Cys Ala Glu Glu Glu Lys Glu 180 185 190Glu Leu3464PRTHomo sapiens 3Met Tyr Ser Asn Val Ile Gly Thr Val Thr Ser Gly Lys Arg Lys Val1 5 10 15Tyr Leu Leu Ser Leu Leu Leu Ile Gly Phe Trp Asp Cys Val Thr Cys 20 25 30His Gly Ser Pro Val Asp Ile Cys Thr Ala Lys Pro Arg Asp Ile Pro 35 40 45Met Asn Pro Met Cys Ile Tyr Arg Ser Pro Glu Lys Lys Ala Thr Glu 50 55 60Asp Glu Gly Ser Glu Gln Lys Ile Pro Glu Ala Thr Asn Arg Arg Val65 70 75 80Trp Glu Leu Ser Lys Ala Asn Ser Arg Phe Ala Thr Thr Phe Tyr Gln 85 90 95His Leu Ala Asp Ser Lys Asn Asp Asn Asp Asn Ile Phe Leu Ser Pro 100 105 110Leu Ser Ile Ser Thr Ala Phe Ala Met Thr Lys Leu Gly Ala Cys Asn 115 120 125Asp Thr Leu Gln Gln Leu Met Glu Val Phe Lys Phe Asp Thr Ile Ser 130 135 140Glu Lys Thr Ser Asp Gln Ile His Phe Phe Phe Ala Lys Leu Asn Cys145 150 155 160Arg Leu Tyr Arg Lys Ala Asn Lys Ser Ser Lys Leu Val Ser Ala Asn 165 170 175Arg Leu Phe Gly Asp Lys Ser Leu Thr Phe Asn Glu Thr Tyr Gln Asp 180 185 190Ile Ser Glu Leu Val Tyr Gly Ala Lys Leu Gln Pro Leu Asp Phe Lys 195 200 205Glu Asn Ala Glu Gln Ser Arg Ala Ala Ile Asn Lys Trp Val Ser Asn 210 215 220Lys Thr Glu Gly Arg Ile Thr Asp Val Ile Pro Ser Glu Ala Ile Asn225 230 235 240Glu Leu Thr Val Leu Val Leu Val Asn Thr Ile Tyr Phe Lys Gly Leu 245 250 255Trp Lys Ser Lys Phe Ser Pro Glu Asn Thr Arg Lys Glu Leu Phe Tyr 260 265 270Lys Ala Asp Gly Glu Ser Cys Ser Ala Ser Met Met Tyr Gln Glu Gly 275 280 285Lys Phe Arg Tyr Arg Arg Val Ala Glu Gly Thr Gln Val Leu Glu Leu 290 295 300Pro Phe Lys Gly Asp Asp Ile Thr Met Val Leu Ile Leu Pro Lys Pro305 310 315 320Glu Lys Ser Leu Ala Lys Val Glu Lys Glu Leu Thr Pro Glu Val Leu 325 330 335Gln Glu Trp Leu Asp Glu Leu Glu Glu Met Met Leu Val Val His Met 340 345 350Pro Arg Phe Arg Ile Glu Asp Gly Phe Ser Leu Lys Glu Gln Leu Gln 355 360 365Asp Met Gly Leu Val Asp Leu Phe Ser Pro Glu Lys Ser Lys Leu Pro 370 375 380Gly Ile Val Ala Glu Gly Arg Asp Asp Leu Tyr Val Ser Asp Ala Phe385 390 395 400His Lys Ala Phe Leu Glu Val Asn Glu Glu Gly Ser Glu Ala Ala Ala 405 410 415Ser Thr Ala Val Val Ile Ala Gly Arg Ser Leu Asn Pro Asn Arg Val 420 425 430Thr Phe Lys Ala Asn Arg Pro Phe Leu Val Phe Ile Arg Glu Val Pro 435 440 445Leu Asn Thr Ile Ile Phe Met Gly Arg Val Ala Asn Pro Cys Val Lys 450 455 46042463PRTHomo sapiens 4His Ile Asn Ile Asp Gln Phe Val Arg Lys Tyr Arg Ala Ala Leu Gly1 5 10 15Lys Leu Pro Gln Gln Ala Asn Asp Tyr Leu Asn Ser Phe Asn Trp Glu 20 25 30Arg Gln Val Ser His Ala Lys Glu Lys Leu Thr Ala Leu Thr Lys Lys 35 40 45Tyr Arg Ile Thr Glu Asn Asp Ile Gln Ile Ala Leu Asp Asp Ala Lys 50 55 60Ile Asn Phe Asn Glu Lys Leu Ser Gln Leu Gln Thr Tyr Met Ile Gln65 70 75 80Phe Asp Gln Tyr Ile Lys Asp Ser Tyr Asp Leu His Asp Leu Lys Ile 85 90 95Ala Ile Ala Asn Ile Ile Asp Glu Ile Ile Glu Lys Leu Lys Ser Leu 100 105 110Asp Glu His Tyr His Ile Arg Val Asn Leu Val Lys Thr Ile His Asp 115 120 125Leu His Leu Phe Ile Glu Asn Ile Asp Phe Asn Lys Ser Gly Ser Ser 130 135 140Thr Ala Ser Trp Ile Gln Asn Val Asp Thr Lys Tyr Gln Ile Arg Ile145 150 155 160Gln Ile Gln Glu Lys Leu Gln Gln Leu Lys Arg His Ile Gln Asn Ile 165 170 175Asp Ile Gln His Leu Ala Gly Lys Leu Lys Gln His Ile Glu Ala Ile 180 185 190Asp Val Arg Val Leu Leu Asp Gln Leu Gly Thr Thr Ile Ser Phe Glu 195 200 205Arg Ile Asn Asp Val Leu Glu His Val Lys His Phe Val Ile Asn Leu 210 215 220Ile Gly Asp Phe Glu Val Ala Glu Lys Ile Asn Ala Phe Arg Ala Lys225 230 235 240Val His Glu Leu Ile Glu Arg Tyr Glu Val Asp Gln Gln Ile Gln Val 245 250 255Leu Met Asp Lys Leu Val Glu Leu Thr His Gln Tyr Lys Leu Lys Glu 260 265 270Thr Ile Gln Lys Leu Ser Asn Val Leu Gln Gln Val Lys Ile Lys Asp 275 280 285Tyr Phe Glu Lys Leu Val Gly Phe Ile Asp Asp Ala Val Lys Lys Leu 290 295 300Asn Glu Leu Ser Phe Lys Thr Phe Ile Glu Asp Val Asn Lys Phe Leu305 310 315 320Asp Met Leu Ile Lys Lys Leu Lys Ser Phe Asp Tyr His Gln Phe Val 325 330 335Asp Glu Thr Asn Asp Lys Ile Arg Glu Val Thr Gln Arg Leu Asn Gly 340 345 350Glu Ile Gln Ala Leu Glu Leu Pro Gln Lys Ala Glu Ala Leu Lys Leu 355 360 365Phe Leu Glu Glu Thr Lys Ala Thr Val Ala Val Tyr Leu Glu Ser Leu 370 375 380Gln Asp Thr Lys Ile Thr Leu Ile Ile Asn Trp Leu Gln Glu Ala Leu385 390 395 400Ser Ser Ala Ser Leu Ala His Met Lys Ala Lys Phe Arg Glu Thr Leu 405 410 415Glu Asp Thr Arg Asp Arg Met Tyr Gln Met Asp Ile Gln Gln Glu Leu 420 425 430Gln Arg Tyr Leu Ser Leu Val Gly Gln Val Tyr Ser Thr Leu Val Thr 435 440 445Tyr Ile Ser Asp Trp Trp Thr Leu Ala Ala Lys Asn Leu Thr Asp Phe 450 455 460Ala Glu Gln Tyr Ser Ile Gln Asp Trp Ala Lys Arg Met Lys Ala Leu465 470 475 480Val Glu Gln Gly Phe Thr Val Pro Glu Ile Lys Thr Ile Leu Gly Thr 485 490 495Met Pro Ala Phe Glu Val Ser Leu Gln Ala Leu Gln Lys Ala Thr Phe 500 505 510Gln Thr Pro Asp Phe Ile Val Pro Leu Thr Asp Leu Arg Ile Pro Ser 515 520 525Val Gln Ile Asn Phe Lys Asp Leu Lys Asn Ile Lys Ile Pro Ser Arg 530 535 540Phe Ser Thr Pro Glu Phe Thr Ile Leu Asn Thr Phe His Ile Pro Ser545 550 555 560Phe Thr Ile Asp Phe Val Glu Met Lys Val Lys Ile Ile Arg Thr Ile 565 570 575Asp Gln Met Gln Asn Ser Glu Leu Gln Trp Pro Val Pro Asp Ile Tyr 580 585 590Leu Arg Asp Leu Lys Val Glu Asp Ile Pro Leu Ala Arg Ile Thr Leu 595 600 605Pro Asp Phe Arg Leu Pro Glu Ile Ala Ile Pro Glu Phe Ile Ile Pro 610 615 620Thr Leu Asn Leu Asn Asp Phe Gln Val Pro Asp Leu His Ile Pro Glu625 630 635 640Phe Gln Leu Pro His Ile Ser His Thr Ile Glu Val Pro Thr Phe Gly 645 650 655Lys Leu Tyr Ser Ile Leu Lys Ile Gln Ser Pro Leu Phe Thr Leu Asp 660 665 670Ala Asn Ala Asp Ile Gly Asn Gly Thr Thr Ser Ala Asn Glu Ala Gly 675 680 685Ile Ala Ala Ser Ile Thr Ala Lys Gly Glu Ser Lys Leu Glu Val Leu 690 695 700Asn Phe Asp Phe Gln Ala Asn Ala Gln Leu Ser Asn Pro Lys Ile Asn705 710 715 720Pro Leu Ala Leu Lys Glu Ser Val Lys Phe Ser Ser Lys Tyr Leu Arg 725 730 735Thr Glu His Gly Ser Glu Met Leu Phe Phe Gly Asn Ala Ile Glu Gly 740 745 750Lys Ser Asn Thr Val Ala Ser Leu His Thr Glu Lys Asn Thr Leu Glu 755 760 765Leu Ser Asn Gly Val Ile Val Lys Ile Asn Asn Gln Leu Thr Leu Asp 770 775 780Ser Asn Thr Lys Tyr Phe His Lys Leu Asn Ile Pro Lys Leu Asp Phe785 790 795 800Ser Ser Gln Ala Asp Leu Arg Asn Glu Ile Lys Thr Leu Leu Lys Ala 805 810 815Gly His Ile Ala Trp Thr Ser Ser Gly Lys Gly Ser Trp Lys Trp Ala 820 825 830Cys Pro Arg Phe Ser Asp Glu Gly Thr His Glu Ser Gln Ile Ser Phe 835 840 845Thr Ile Glu Gly Pro Leu Thr Ser Phe Gly Leu Ser Asn Lys Ile Asn 850 855 860Ser Lys His Leu Arg Val Asn Gln Asn Leu Val Tyr Glu Ser Gly Ser865 870 875 880Leu Asn Phe Ser Lys Leu Glu Ile Gln Ser Gln Val Asp Ser Gln His 885 890 895Val Gly His Ser Val Leu Thr Ala Lys Gly Met Ala Leu Phe Gly Glu 900 905 910Gly Lys Ala Glu Phe Thr Gly Arg His Asp Ala His Leu Asn Gly Lys 915 920 925Val Ile Gly Thr Leu Lys Asn Ser Leu Phe Phe Ser Ala Gln Pro Phe 930 935 940Glu Ile Thr Ala Ser Thr Asn Asn Glu Gly Asn Leu Lys Val Arg Phe945 950 955 960Pro Leu Arg Leu Thr Gly Lys Ile Asp Phe Leu Asn Asn Tyr Ala Leu 965 970 975Phe Leu Ser Pro Ser Ala Gln Gln Ala Ser Trp Gln Val Ser Ala Arg 980 985 990Phe Asn Gln Tyr Lys Tyr Asn Gln Asn Phe Ser Ala Gly Asn Asn Glu 995 1000 1005Asn Ile Met Glu Ala His Val Gly Ile Asn Gly Glu Ala Asn Leu 1010 1015 1020Asp Phe Leu Asn Ile Pro Leu Thr Ile Pro Glu Met Arg Leu Pro 1025 1030 1035Tyr Thr Ile Ile Thr Thr Pro Pro Leu Lys Asp Phe Ser Leu Trp 1040 1045 1050Glu Lys Thr Gly Leu Lys Glu Phe Leu Lys Thr Thr Lys Gln Ser 1055 1060 1065Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys Lys Asn Lys His Arg 1070 1075 1080His Ser Ile Thr Asn Pro Leu Ala Val Leu Cys Glu Phe Ile Ser 1085 1090 1095Gln Ser Ile Lys Ser Phe Asp Arg His Phe Glu Lys Asn Arg Asn 1100 1105 1110Asn Ala Leu Asp Phe Val Thr Lys Ser Tyr Asn Glu Thr Lys Ile 1115 1120 1125Lys Phe Asp Lys Tyr Lys Ala Glu Lys Ser His Asp Glu Leu Pro 1130 1135 1140Arg Thr Phe Gln Ile Pro Gly Tyr Thr Val Pro Val Val Asn Val 1145 1150 1155Glu Val Ser Pro Phe Thr Ile Glu Met Ser Ala Phe Gly Tyr Val 1160 1165 1170Phe Pro Lys Ala Val Ser Met Pro Ser Phe Ser Ile Leu Gly Ser 1175 1180 1185Asp Val Arg Val Pro Ser Tyr Thr Leu Ile Leu Pro Ser Leu Glu 1190 1195 1200Leu Pro Val Leu His Val Pro Arg Asn Leu Lys Leu Ser Leu Pro 1205 1210 1215His Phe Lys Glu Leu Cys Thr Ile Ser His Ile Phe Ile Pro Ala 1220 1225 1230Met Gly Asn Ile Thr Tyr Asp Phe Ser Phe Lys Ser Ser Val Ile 1235 1240 1245Thr Leu Asn Thr Asn Ala Glu Leu Phe Asn Gln Ser Asp Ile Val 1250 1255 1260Ala His Leu Leu Ser Ser Ser Ser Ser Val Ile Asp Ala Leu Gln 1265 1270 1275Tyr Lys Leu Glu Gly Thr Thr Arg Leu Thr Arg Lys Arg Gly Leu 1280 1285 1290Lys Leu Ala Thr Ala Leu Ser Leu Ser Asn Lys Phe Val Glu Gly 1295 1300 1305Ser His Asn Ser Thr Val Ser Leu Thr Thr Lys Asn Met Glu Val 1310 1315 1320Ser Val Ala Lys Thr Thr Lys Ala Glu Ile Pro Ile Leu Arg Met 1325 1330 1335Asn Phe Lys Gln Glu Leu Asn Gly Asn Thr Lys Ser Lys Pro Thr 1340 1345 1350Val Ser Ser Ser Met Glu Phe Lys Tyr Asp Phe Asn Ser Ser Met 1355 1360 1365Leu Tyr Ser Thr Ala Lys Gly Ala Val Asp His Lys Leu Ser Leu 1370 1375 1380Glu Ser Leu Thr Ser Tyr Phe Ser Ile Glu Ser Ser Thr Lys Gly 1385 1390 1395Asp Val Lys Gly Ser Val Leu Ser Arg Glu Tyr Ser Gly Thr Ile 1400 1405 1410Ala Ser Glu Ala Asn Thr Tyr Leu Asn Ser Lys Ser Thr Arg Ser 1415 1420 1425Ser Val Lys Leu Gln Gly Thr Ser Lys Ile Asp Asp Ile Trp Asn 1430 1435 1440Leu Glu Val Lys Glu Asn Phe Ala Gly Glu Ala Thr Leu Gln Arg 1445 1450 1455Ile Tyr Ser Leu Trp Glu His Ser Thr Lys Asn His Leu Gln Leu 1460 1465 1470Glu Gly Leu Phe Phe Thr Asn Gly Glu His Thr Ser Lys Ala Thr 1475 1480 1485Leu Glu Leu Ser Pro Trp Gln Met Ser Ala Leu Val Gln Val His 1490 1495 1500Ala Ser Gln Pro Ser Ser Phe His Asp Phe Pro Asp Leu Gly Gln 1505 1510 1515Glu Val Ala Leu Asn Ala Asn Thr Lys Asn Gln Lys Ile Arg Trp 1520 1525 1530Lys Asn Glu Val Arg Ile His Ser Gly Ser Phe Gln Ser Gln Val 1535 1540 1545Glu Leu Ser Asn Asp Gln Glu Lys Ala His Leu Asp Ile Ala Gly 1550 1555 1560Ser Leu Glu Gly His Leu Arg Phe Leu Lys Asn Ile Ile Leu Pro 1565 1570 1575Val Tyr Asp Lys Ser Leu Trp Asp Phe Leu Lys Leu Asp Val Thr 1580 1585 1590Thr Ser Ile Gly Arg Arg Gln His Leu Arg Val Ser Thr Ala Phe 1595

1600 1605Val Tyr Thr Lys Asn Pro Asn Gly Tyr Ser Phe Ser Ile Pro Val 1610 1615 1620Lys Val Leu Ala Asp Lys Phe Ile Thr Pro Gly Leu Lys Leu Asn 1625 1630 1635Asp Leu Asn Ser Val Leu Val Met Pro Thr Phe His Val Pro Phe 1640 1645 1650Thr Asp Leu Gln Val Pro Ser Cys Lys Leu Asp Phe Arg Glu Ile 1655 1660 1665Gln Ile Tyr Lys Lys Leu Arg Thr Ser Ser Phe Ala Leu Asn Leu 1670 1675 1680Pro Thr Leu Pro Glu Val Lys Phe Pro Glu Val Asp Val Leu Thr 1685 1690 1695Lys Tyr Ser Gln Pro Glu Asp Ser Leu Ile Pro Phe Phe Glu Ile 1700 1705 1710Thr Val Pro Glu Ser Gln Leu Thr Val Ser Gln Phe Thr Leu Pro 1715 1720 1725Lys Ser Val Ser Asp Gly Ile Ala Ala Leu Asp Leu Asn Ala Val 1730 1735 1740Ala Asn Lys Ile Ala Asp Phe Glu Leu Pro Thr Ile Ile Val Pro 1745 1750 1755Glu Gln Thr Ile Glu Ile Pro Ser Ile Lys Phe Ser Val Pro Ala 1760 1765 1770Gly Ile Val Ile Pro Ser Phe Gln Ala Leu Thr Ala Arg Phe Glu 1775 1780 1785Val Asp Ser Pro Val Tyr Asn Ala Thr Trp Ser Ala Ser Leu Lys 1790 1795 1800Asn Lys Ala Asp Tyr Val Glu Thr Val Leu Asp Ser Thr Cys Ser 1805 1810 1815Ser Thr Val Gln Phe Leu Glu Tyr Glu Leu Asn Val Leu Gly Thr 1820 1825 1830His Lys Ile Glu Asp Gly Thr Leu Ala Ser Lys Thr Lys Gly Thr 1835 1840 1845Leu Ala His Arg Asp Phe Ser Ala Glu Tyr Glu Glu Asp Gly Lys 1850 1855 1860Phe Glu Gly Leu Gln Glu Trp Glu Gly Lys Ala His Leu Asn Ile 1865 1870 1875Lys Ser Pro Ala Phe Thr Asp Leu His Leu Arg Tyr Gln Lys Asp 1880 1885 1890Lys Lys Gly Ile Ser Thr Ser Ala Ala Ser Pro Ala Val Gly Thr 1895 1900 1905Val Gly Met Asp Met Asp Glu Asp Asp Asp Phe Ser Lys Trp Asn 1910 1915 1920Phe Tyr Tyr Ser Pro Gln Ser Ser Pro Asp Lys Lys Leu Thr Ile 1925 1930 1935Phe Lys Thr Glu Leu Arg Val Arg Glu Ser Asp Glu Glu Thr Gln 1940 1945 1950Ile Lys Val Asn Trp Glu Glu Glu Ala Ala Ser Gly Leu Leu Thr 1955 1960 1965Ser Leu Lys Asp Asn Val Pro Lys Ala Thr Gly Val Leu Tyr Asp 1970 1975 1980Tyr Val Asn Lys Tyr His Trp Glu His Thr Gly Leu Thr Leu Arg 1985 1990 1995Glu Val Ser Ser Lys Leu Arg Arg Asn Leu Gln Asn Asn Ala Glu 2000 2005 2010Trp Val Tyr Gln Gly Ala Ile Arg Gln Ile Asp Asp Ile Asp Val 2015 2020 2025Arg Phe Gln Lys Ala Ala Ser Gly Thr Thr Gly Thr Tyr Gln Glu 2030 2035 2040Trp Lys Asp Lys Ala Gln Asn Leu Tyr Gln Glu Leu Leu Thr Gln 2045 2050 2055Glu Gly Gln Ala Ser Phe Gln Gly Leu Lys Asp Asn Val Phe Asp 2060 2065 2070Gly Leu Val Arg Val Thr Gln Lys Phe His Met Lys Val Lys His 2075 2080 2085Leu Ile Asp Ser Leu Ile Asp Phe Leu Asn Phe Pro Arg Phe Gln 2090 2095 2100Phe Pro Gly Lys Pro Gly Ile Tyr Thr Arg Glu Glu Leu Cys Thr 2105 2110 2115Met Phe Ile Arg Glu Val Gly Thr Val Leu Ser Gln Val Tyr Ser 2120 2125 2130Lys Val His Asn Gly Ser Glu Ile Leu Phe Ser Tyr Phe Gln Asp 2135 2140 2145Leu Val Ile Thr Leu Pro Phe Glu Leu Arg Lys His Lys Leu Ile 2150 2155 2160Asp Val Ile Ser Met Tyr Arg Glu Leu Leu Lys Asp Leu Ser Lys 2165 2170 2175Glu Ala Gln Glu Val Phe Lys Ala Ile Gln Ser Leu Lys Thr Thr 2180 2185 2190Glu Val Leu Arg Asn Leu Gln Asp Leu Leu Gln Phe Ile Phe Gln 2195 2200 2205Leu Ile Glu Asp Asn Ile Lys Gln Leu Lys Glu Met Lys Phe Thr 2210 2215 2220Tyr Leu Ile Asn Tyr Ile Gln Asp Glu Ile Asn Thr Ile Phe Asn 2225 2230 2235Asp Tyr Ile Pro Tyr Val Phe Lys Leu Leu Lys Glu Asn Leu Cys 2240 2245 2250Leu Asn Leu His Lys Phe Asn Glu Phe Ile Gln Asn Glu Leu Gln 2255 2260 2265Glu Ala Ser Gln Glu Leu Gln Gln Ile His Gln Tyr Ile Met Ala 2270 2275 2280Leu Arg Glu Glu Tyr Phe Asp Pro Ser Ile Val Gly Trp Thr Val 2285 2290 2295Lys Tyr Tyr Glu Leu Glu Glu Lys Ile Val Ser Leu Ile Lys Asn 2300 2305 2310Leu Leu Val Ala Leu Lys Asp Phe His Ser Glu Tyr Ile Val Ser 2315 2320 2325Ala Ser Asn Phe Thr Ser Gln Leu Ser Ser Gln Val Glu Gln Phe 2330 2335 2340Leu His Arg Asn Ile Gln Glu Tyr Leu Ser Ile Leu Thr Asp Pro 2345 2350 2355Asp Gly Lys Gly Lys Glu Lys Ile Ala Glu Leu Ser Ala Thr Ala 2360 2365 2370Gln Glu Ile Ile Lys Ser Gln Ala Ile Ala Thr Lys Lys Ile Ile 2375 2380 2385Ser Asp Tyr His Gln Gln Phe Arg Tyr Lys Leu Gln Asp Phe Ser 2390 2395 2400Asp Gln Leu Ser Asp Tyr Tyr Glu Lys Phe Ile Ala Glu Ser Lys 2405 2410 2415Arg Leu Ile Asp Leu Ser Ile Gln Asn Tyr His Thr Phe Leu Ile 2420 2425 2430Tyr Ile Thr Glu Leu Leu Lys Lys Leu Gln Ser Thr Thr Val Met 2435 2440 2445Asn Pro Tyr Met Lys Leu Ala Pro Gly Glu Leu Thr Ile Ile Leu 2450 2455 24605474PRTHomo sapiens 5Met Lys Arg Val Leu Val Leu Leu Leu Ala Val Ala Phe Gly His Ala1 5 10 15Leu Glu Arg Gly Arg Asp Tyr Glu Lys Asn Lys Val Cys Lys Glu Phe 20 25 30Ser His Leu Gly Lys Glu Asp Phe Thr Ser Leu Ser Leu Val Leu Tyr 35 40 45Ser Arg Lys Phe Pro Ser Gly Thr Phe Glu Gln Val Ser Gln Leu Val 50 55 60Lys Glu Val Val Ser Leu Thr Glu Ala Cys Cys Ala Glu Gly Ala Asp65 70 75 80Pro Asp Cys Tyr Asp Thr Arg Thr Ser Ala Leu Ser Ala Lys Ser Cys 85 90 95Glu Ser Asn Ser Pro Phe Pro Val His Pro Gly Thr Ala Glu Cys Cys 100 105 110Thr Lys Glu Gly Leu Glu Arg Lys Leu Cys Met Ala Ala Leu Lys His 115 120 125Gln Pro Gln Glu Phe Pro Thr Tyr Val Glu Pro Thr Asn Asp Glu Ile 130 135 140Cys Glu Ala Phe Arg Lys Asp Pro Lys Glu Tyr Ala Asn Gln Phe Met145 150 155 160Trp Glu Tyr Ser Thr Asn Tyr Glu Gln Ala Pro Leu Ser Leu Leu Val 165 170 175Ser Tyr Thr Lys Ser Tyr Leu Ser Met Val Gly Ser Cys Cys Thr Ser 180 185 190Ala Ser Pro Thr Val Cys Phe Leu Lys Glu Arg Leu Gln Leu Lys His 195 200 205Leu Ser Leu Leu Thr Thr Leu Ser Asn Arg Val Cys Ser Gln Tyr Ala 210 215 220Ala Tyr Gly Glu Lys Lys Ser Arg Leu Ser Asn Leu Ile Lys Leu Ala225 230 235 240Gln Lys Val Pro Thr Ala Asp Leu Glu Asp Val Leu Pro Leu Ala Glu 245 250 255Asp Ile Thr Asn Ile Leu Ser Lys Cys Cys Glu Ser Ala Ser Glu Asp 260 265 270Cys Met Ala Lys Glu Leu Pro Glu His Thr Val Lys Leu Cys Asp Asn 275 280 285Leu Ser Thr Lys Asn Ser Lys Phe Glu Asp Cys Cys Gln Glu Lys Thr 290 295 300Ala Met Asp Val Phe Val Cys Thr Tyr Phe Met Pro Ala Ala Gln Leu305 310 315 320Pro Glu Leu Pro Asp Val Arg Leu Pro Thr Asn Lys Asp Val Cys Asp 325 330 335Pro Gly Asn Thr Lys Val Met Asp Lys Tyr Thr Phe Glu Leu Ser Arg 340 345 350Arg Thr His Leu Pro Glu Val Phe Leu Ser Lys Val Leu Glu Pro Thr 355 360 365Leu Lys Ser Leu Gly Glu Cys Cys Asp Val Glu Asp Ser Thr Thr Cys 370 375 380Phe Asn Ala Lys Gly Pro Leu Leu Lys Lys Glu Leu Ser Ser Phe Ile385 390 395 400Asp Lys Gly Gln Glu Leu Cys Ala Asp Tyr Ser Glu Asn Thr Phe Thr 405 410 415Glu Tyr Lys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Glu 420 425 430Ala Thr Pro Thr Glu Leu Ala Lys Leu Val Asn Lys Arg Ser Asp Phe 435 440 445Ala Ser Asn Cys Cys Ser Ile Asn Ser Pro Pro Leu Tyr Cys Asp Ser 450 455 460Glu Ile Asp Ala Glu Leu Lys Asn Ile Leu465 4706474PRTHomo sapiens 6Met Lys Arg Val Leu Val Leu Leu Leu Ala Val Ala Phe Gly His Ala1 5 10 15Leu Glu Arg Gly Arg Asp Tyr Glu Lys Asn Lys Val Cys Lys Glu Phe 20 25 30Ser His Leu Gly Lys Glu Asp Phe Thr Ser Leu Ser Leu Val Leu Tyr 35 40 45Ser Arg Lys Phe Pro Ser Gly Thr Phe Glu Gln Val Ser Gln Leu Val 50 55 60Lys Glu Val Val Ser Leu Thr Glu Ala Cys Cys Ala Glu Gly Ala Asp65 70 75 80Pro Asp Cys Tyr Asp Thr Arg Thr Ser Ala Leu Ser Ala Lys Ser Cys 85 90 95Glu Ser Asn Ser Pro Phe Pro Val His Pro Gly Thr Ala Glu Cys Cys 100 105 110Thr Lys Glu Gly Leu Glu Arg Lys Leu Cys Met Ala Ala Leu Lys His 115 120 125Gln Pro Gln Glu Phe Pro Thr Tyr Val Glu Pro Thr Asn Asp Glu Ile 130 135 140Cys Glu Ala Phe Arg Lys Asp Pro Lys Glu Tyr Ala Asn Gln Phe Met145 150 155 160Trp Glu Tyr Ser Thr Asn Tyr Gly Gln Ala Pro Leu Ser Leu Leu Val 165 170 175Ser Tyr Thr Lys Ser Tyr Leu Ser Met Val Gly Ser Cys Cys Thr Ser 180 185 190Ala Ser Pro Thr Val Cys Phe Leu Lys Glu Arg Leu Gln Leu Lys His 195 200 205Leu Ser Leu Leu Thr Thr Leu Ser Asn Arg Val Cys Ser Gln Tyr Ala 210 215 220Ala Tyr Gly Glu Lys Lys Ser Arg Leu Ser Asn Leu Ile Lys Leu Ala225 230 235 240Gln Lys Val Pro Thr Ala Asp Leu Glu Asp Val Leu Pro Leu Ala Glu 245 250 255Asp Ile Thr Asn Ile Leu Ser Lys Cys Cys Glu Ser Ala Ser Glu Asp 260 265 270Cys Met Ala Lys Glu Leu Pro Glu His Thr Val Lys Leu Cys Asp Asn 275 280 285Leu Ser Thr Lys Asn Ser Lys Phe Glu Asp Cys Cys Gln Glu Lys Thr 290 295 300Ala Met Asp Val Phe Val Cys Thr Tyr Phe Met Pro Ala Ala Gln Leu305 310 315 320Pro Glu Leu Pro Asp Val Glu Leu Pro Thr Asn Lys Asp Val Cys Asp 325 330 335Pro Gly Asn Thr Lys Val Met Asp Lys Tyr Thr Phe Glu Leu Ser Arg 340 345 350Arg Thr His Leu Pro Glu Val Phe Leu Ser Lys Val Leu Glu Pro Thr 355 360 365Leu Lys Ser Leu Gly Glu Cys Cys Asp Val Glu Asp Ser Thr Thr Cys 370 375 380Phe Asn Ala Lys Gly Pro Leu Leu Lys Lys Glu Leu Ser Ser Phe Ile385 390 395 400Asp Lys Gly Gln Glu Leu Cys Ala Asp Tyr Ser Glu Asn Thr Phe Thr 405 410 415Glu Tyr Lys Lys Lys Leu Ala Glu Arg Leu Lys Ala Lys Leu Pro Glu 420 425 430Ala Thr Pro Thr Glu Leu Ala Lys Leu Val Asn Lys Arg Ser Asp Phe 435 440 445Ala Ser Asn Cys Cys Ser Ile Asn Ser Pro Pro Leu Tyr Cys Asp Ser 450 455 460Glu Ile Asp Ala Glu Leu Lys Asn Ile Leu465 4707318PRTHomo sapiens 7Met Ala Ser Pro Gly Cys Leu Leu Cys Val Leu Gly Leu Leu Leu Cys1 5 10 15Gly Ala Ala Ser Leu Glu Leu Ser Arg Pro His Gly Asp Thr Ala Lys 20 25 30Lys Pro Ile Ile Gly Ile Leu Met Gln Lys Cys Arg Asn Lys Val Met 35 40 45Lys Asn Tyr Gly Arg Tyr Tyr Ile Ala Ala Ser Tyr Val Lys Tyr Leu 50 55 60Glu Ser Ala Gly Ala Arg Val Val Pro Val Arg Leu Asp Leu Thr Glu65 70 75 80Lys Asp Tyr Glu Ile Leu Phe Lys Ser Ile Asn Gly Ile Leu Phe Pro 85 90 95Gly Gly Ser Val Asp Leu Arg Arg Ser Asp Tyr Ala Lys Val Ala Lys 100 105 110Ile Phe Tyr Asn Leu Ser Ile Gln Ser Phe Asp Asp Gly Asp Tyr Phe 115 120 125Pro Val Trp Gly Thr Cys Leu Gly Phe Glu Glu Leu Ser Leu Leu Ile 130 135 140Ser Gly Glu Cys Leu Leu Thr Ala Thr Asp Thr Val Asp Val Ala Met145 150 155 160Pro Leu Asn Phe Thr Gly Gly Gln Leu His Ser Arg Met Phe Gln Asn 165 170 175Phe Pro Thr Glu Leu Leu Leu Ser Leu Ala Val Glu Pro Leu Thr Ala 180 185 190Asn Phe His Lys Trp Ser Leu Ser Val Lys Asn Phe Thr Met Asn Glu 195 200 205Lys Leu Lys Lys Phe Phe Asn Val Leu Thr Thr Asn Thr Asp Gly Lys 210 215 220Ile Glu Phe Ile Ser Thr Met Glu Gly Tyr Lys Tyr Pro Val Tyr Gly225 230 235 240Val Gln Trp His Pro Glu Lys Ala Pro Tyr Glu Trp Lys Asn Leu Asp 245 250 255Gly Ile Ser His Ala Pro Asn Ala Val Lys Thr Ala Phe Tyr Leu Ala 260 265 270Glu Phe Phe Val Asn Glu Ala Arg Lys Asn Asn His His Phe Lys Ser 275 280 285Glu Ser Glu Glu Glu Lys Ala Leu Ile Tyr Gln Phe Ser Pro Ile Tyr 290 295 300Thr Gly Asn Ile Ser Ser Phe Gln Gln Cys Tyr Ile Phe Asp305 310 3158709PRTHomo sapiens 8Met Ala Thr Ala Gly Gly Gly Ser Gly Ala Asp Pro Gly Ser Arg Gly1 5 10 15Leu Leu Arg Leu Leu Ser Phe Cys Val Leu Leu Ala Gly Leu Cys Arg 20 25 30Gly Asn Ser Val Glu Arg Lys Ile Tyr Ile Pro Leu Asn Lys Thr Ala 35 40 45Pro Cys Val Arg Leu Leu Asn Ala Thr His Gln Ile Gly Cys Gln Ser 50 55 60Ser Ile Ser Gly Asp Thr Gly Val Ile His Val Val Glu Lys Glu Glu65 70 75 80Asp Leu Gln Trp Val Leu Thr Asp Gly Pro Asn Pro Pro Tyr Met Val 85 90 95Leu Leu Glu Ser Lys His Phe Thr Arg Asp Leu Met Glu Lys Leu Lys 100 105 110Gly Arg Thr Ser Arg Ile Ala Gly Leu Ala Val Ser Leu Thr Lys Pro 115 120 125Ser Pro Ala Ser Gly Phe Ser Pro Ser Val Gln Cys Pro Asn Asp Gly 130 135 140Phe Gly Val Tyr Ser Asn Ser Tyr Gly Pro Glu Phe Ala His Cys Arg145 150 155 160Glu Ile Gln Trp Asn Ser Leu Gly Asn Gly Leu Ala Tyr Glu Asp Phe 165 170 175Ser Phe Pro Ile Phe Leu Leu Glu Asp Glu Asn Glu Thr Lys Val Ile 180 185 190Lys Gln Cys Tyr Gln Asp His Asn Leu Ser Gln Asn Gly Ser Ala Pro 195 200 205Thr Phe Pro Leu Cys Ala Met Gln Leu Phe Ser His Met His Ala Val 210 215 220Ile Ser Thr Ala Thr Cys Met Arg Arg Ser Ser Ile Gln Ser Thr Phe225 230 235 240Ser Ile Asn Pro Glu Ile Val Cys Asp Pro Leu Ser Asp Tyr Asn Val 245 250 255Trp Ser Met Leu Lys Pro Ile Asn Thr Thr Gly Thr Leu Lys Pro Asp 260 265 270Asp Arg Val Val Val Ala Ala Thr Arg Leu Asp Ser Arg Ser Phe Phe 275 280 285Trp Asn Val Ala Pro Gly Ala Glu Ser Ala Val Ala Ser Phe Val Thr 290 295 300Gln Leu Ala Ala Ala Glu Ala Leu Gln Lys Ala Pro Asp Val Thr Thr305 310 315 320Leu Pro Arg Asn Val Met Phe Val Phe Phe Gln Gly Glu Thr Phe Asp 325 330

335Tyr Ile Gly Ser Ser Arg Met Val Tyr Asp Met Glu Lys Gly Lys Phe 340 345 350Pro Val Gln Leu Glu Asn Val Asp Ser Phe Val Glu Leu Gly Gln Val 355 360 365Ala Leu Arg Thr Ser Leu Glu Leu Trp Met His Thr Asp Pro Val Ser 370 375 380Gln Lys Asn Glu Ser Val Arg Asn Gln Val Glu Asp Leu Leu Ala Thr385 390 395 400Leu Glu Lys Ser Gly Ala Gly Val Pro Ala Val Ile Leu Arg Arg Pro 405 410 415Asn Gln Ser Gln Pro Leu Pro Pro Ser Ser Leu Gln Arg Phe Leu Arg 420 425 430Ala Arg Asn Ile Ser Gly Val Val Leu Ala Asp His Ser Gly Ala Phe 435 440 445His Asn Lys Tyr Tyr Gln Ser Ile Tyr Asp Thr Ala Glu Asn Ile Asn 450 455 460Val Ser Tyr Pro Glu Trp Leu Ser Pro Glu Glu Asp Leu Asn Phe Val465 470 475 480Thr Asp Thr Ala Lys Ala Leu Ala Asp Val Ala Thr Val Leu Gly Arg 485 490 495Ala Leu Tyr Glu Leu Ala Gly Gly Thr Asn Phe Ser Asp Thr Val Gln 500 505 510Ala Asp Pro Gln Thr Val Thr Arg Leu Leu Tyr Gly Phe Leu Ile Lys 515 520 525Ala Asn Asn Ser Trp Phe Gln Ser Ile Leu Arg Gln Asp Leu Arg Ser 530 535 540Tyr Leu Gly Asp Gly Pro Leu Gln His Tyr Ile Ala Val Ser Ser Pro545 550 555 560Thr Asn Thr Thr Tyr Val Val Gln Tyr Ala Leu Ala Asn Leu Thr Gly 565 570 575Thr Val Val Asn Leu Thr Arg Glu Gln Cys Gln Asp Pro Ser Lys Val 580 585 590Pro Ser Glu Asn Lys Asp Leu Tyr Glu Tyr Ser Trp Val Gln Gly Pro 595 600 605Leu His Ser Asn Glu Thr Asp Arg Leu Pro Arg Cys Val Arg Ser Thr 610 615 620Ala Arg Leu Ala Arg Ala Leu Ser Pro Ala Phe Glu Leu Ser Gln Trp625 630 635 640Ser Ser Thr Glu Tyr Ser Thr Trp Thr Glu Ser Arg Trp Lys Asp Ile 645 650 655Arg Ala Arg Ile Phe Leu Ile Ala Ser Lys Glu Leu Glu Leu Ile Thr 660 665 670Leu Thr Val Gly Phe Gly Ile Leu Ile Phe Ser Leu Ile Val Thr Tyr 675 680 685Cys Ile Asn Ala Lys Ala Asp Val Leu Phe Ile Ala Pro Arg Glu Pro 690 695 700Gly Ala Val Ser Tyr7059347PRTHomo sapiens 9Met Ala Gln Ser Arg Asp Gly Gly Asn Pro Phe Ala Glu Pro Ser Glu1 5 10 15Leu Asp Asn Pro Phe Gln Asp Pro Ala Val Ile Gln His Arg Pro Ser 20 25 30Arg Gln Tyr Ala Thr Leu Asp Val Tyr Asn Pro Phe Glu Thr Arg Glu 35 40 45Pro Pro Pro Ala Tyr Glu Pro Pro Ala Pro Ala Pro Leu Pro Pro Pro 50 55 60Ser Ala Pro Ser Leu Gln Pro Ser Arg Lys Leu Ser Pro Thr Glu Pro65 70 75 80Lys Asn Tyr Gly Ser Tyr Ser Thr Gln Ala Ser Ala Ala Ala Ala Thr 85 90 95Ala Glu Leu Leu Lys Lys Gln Glu Glu Leu Asn Arg Lys Ala Glu Glu 100 105 110Leu Asp Arg Arg Glu Arg Glu Leu Gln His Ala Ala Leu Gly Gly Thr 115 120 125Ala Thr Arg Gln Asn Asn Trp Pro Pro Leu Pro Ser Phe Cys Pro Val 130 135 140Gln Pro Cys Phe Phe Gln Asp Ile Ser Met Glu Ile Pro Gln Glu Phe145 150 155 160Gln Lys Thr Val Ser Thr Met Tyr Tyr Leu Trp Met Cys Ser Thr Leu 165 170 175Ala Leu Leu Leu Asn Phe Leu Ala Cys Leu Ala Ser Phe Cys Val Glu 180 185 190Thr Asn Asn Gly Ala Gly Phe Gly Leu Ser Ile Leu Trp Val Leu Leu 195 200 205Phe Thr Pro Cys Ser Phe Val Cys Trp Tyr Arg Pro Met Tyr Lys Ala 210 215 220Phe Arg Ser Asp Ser Ser Phe Asn Phe Phe Val Phe Phe Phe Ile Phe225 230 235 240Phe Val Gln Asp Val Leu Phe Val Leu Gln Ala Ile Gly Ile Pro Gly 245 250 255Trp Gly Phe Ser Gly Trp Ile Ser Ala Leu Val Val Pro Lys Gly Asn 260 265 270Thr Ala Val Ser Val Leu Met Leu Leu Val Ala Leu Leu Phe Thr Gly 275 280 285Ile Ala Val Leu Gly Ile Val Met Leu Lys Arg Ile His Ser Leu Tyr 290 295 300Arg Arg Thr Gly Ala Ser Phe Gln Lys Ala Gln Gln Glu Phe Ala Ala305 310 315 320Gly Val Phe Ser Asn Pro Ala Val Arg Thr Ala Ala Ala Asn Ala Ala 325 330 335Ala Gly Ala Ala Glu Asn Ala Phe Arg Ala Pro 340 34510321PRTHomo sapiens 10Met Ala Gln Ser Arg Asp Gly Gly Asn Pro Phe Ala Glu Pro Ser Glu1 5 10 15Leu Asp Asn Pro Phe Gln Pro Pro Pro Ala Tyr Glu Pro Pro Ala Pro 20 25 30Ala Pro Leu Pro Pro Pro Ser Ala Pro Ser Leu Gln Pro Ser Arg Lys 35 40 45Leu Ser Pro Thr Glu Pro Lys Asn Tyr Gly Ser Tyr Ser Thr Gln Ala 50 55 60Ser Ala Ala Ala Ala Thr Ala Glu Leu Leu Lys Lys Gln Glu Glu Leu65 70 75 80Asn Arg Lys Ala Glu Glu Leu Asp Arg Arg Glu Arg Glu Leu Gln His 85 90 95Ala Ala Leu Gly Gly Thr Ala Thr Arg Gln Asn Asn Trp Pro Pro Leu 100 105 110Pro Ser Phe Cys Pro Val Gln Pro Cys Phe Phe Gln Asp Ile Ser Met 115 120 125Glu Ile Pro Gln Glu Phe Gln Lys Thr Val Ser Thr Met Tyr Tyr Leu 130 135 140Trp Met Cys Ser Thr Leu Ala Leu Leu Leu Asn Phe Leu Ala Cys Leu145 150 155 160Ala Ser Phe Cys Val Glu Thr Asn Asn Gly Ala Gly Phe Gly Leu Ser 165 170 175Ile Leu Trp Val Leu Leu Phe Thr Pro Cys Ser Phe Val Cys Trp Tyr 180 185 190Arg Pro Met Tyr Lys Ala Phe Arg Ser Asp Ser Ser Phe Asn Phe Phe 195 200 205Val Phe Phe Phe Ile Phe Phe Val Gln Asp Val Leu Phe Val Leu Gln 210 215 220Ala Ile Gly Ile Pro Gly Trp Gly Phe Ser Gly Trp Ile Ser Ala Leu225 230 235 240Val Val Pro Lys Gly Asn Thr Ala Val Ser Val Leu Met Leu Leu Val 245 250 255Ala Leu Leu Phe Thr Gly Ile Ala Val Leu Gly Ile Val Met Leu Lys 260 265 270Arg Ile His Ser Leu Tyr Arg Arg Thr Gly Ala Ser Phe Gln Lys Ala 275 280 285Gln Gln Glu Phe Ala Ala Gly Val Phe Ser Asn Pro Ala Val Arg Thr 290 295 300Ala Ala Ala Asn Ala Ala Ala Gly Ala Ala Glu Asn Ala Phe Arg Ala305 310 315 320Pro11541PRTHomo sapiens 11Met Lys Phe Leu Ile Phe Ala Phe Phe Gly Gly Val His Leu Leu Ser1 5 10 15Leu Cys Ser Gly Lys Ala Ile Cys Lys Asn Gly Ile Ser Lys Arg Thr 20 25 30Phe Glu Glu Ile Lys Glu Glu Ile Ala Ser Cys Gly Asp Val Ala Lys 35 40 45Ala Ile Ile Asn Leu Ala Val Tyr Gly Lys Ala Gln Asn Arg Ser Tyr 50 55 60Glu Arg Leu Ala Leu Leu Val Asp Thr Val Gly Pro Arg Leu Ser Gly65 70 75 80Ser Lys Asn Leu Glu Lys Ala Ile Gln Ile Met Tyr Gln Asn Leu Gln 85 90 95Gln Asp Gly Leu Glu Lys Val His Leu Glu Pro Val Arg Ile Pro His 100 105 110Trp Glu Arg Gly Glu Glu Ser Ala Val Met Leu Glu Pro Arg Ile His 115 120 125Lys Ile Ala Ile Leu Gly Leu Gly Ser Ser Ile Gly Thr Pro Pro Glu 130 135 140Gly Ile Thr Ala Glu Val Leu Val Val Thr Ser Phe Asp Glu Leu Gln145 150 155 160Arg Arg Ala Ser Glu Ala Arg Gly Lys Ile Val Val Tyr Asn Gln Pro 165 170 175Tyr Ile Asn Tyr Ser Arg Thr Val Gln Tyr Arg Thr Gln Gly Ala Val 180 185 190Glu Ala Ala Lys Val Gly Ala Leu Ala Ser Leu Ile Arg Ser Val Ala 195 200 205Ser Phe Ser Ile Tyr Ser Pro His Thr Gly Ile Gln Glu Tyr Gln Asp 210 215 220Gly Val Pro Lys Ile Pro Thr Ala Cys Ile Thr Val Glu Asp Ala Glu225 230 235 240Met Met Ser Arg Met Ala Ser His Gly Ile Lys Ile Val Ile Gln Leu 245 250 255Lys Met Gly Ala Lys Thr Tyr Pro Asp Thr Asp Ser Phe Asn Thr Val 260 265 270Ala Glu Ile Thr Gly Ser Lys Tyr Pro Glu Gln Val Val Leu Val Ser 275 280 285Gly His Leu Asp Ser Trp Asp Val Gly Gln Gly Ala Met Asp Asp Gly 290 295 300Gly Gly Ala Phe Ile Ser Trp Glu Ala Leu Ser Leu Ile Lys Asp Leu305 310 315 320Gly Leu Arg Pro Lys Arg Thr Leu Arg Leu Val Leu Trp Thr Ala Glu 325 330 335Glu Gln Gly Gly Val Gly Ala Phe Gln Tyr Tyr Gln Leu His Lys Val 340 345 350Asn Ile Ser Asn Tyr Ser Leu Val Met Glu Ser Asp Ala Gly Thr Phe 355 360 365Leu Pro Thr Gly Leu Gln Phe Thr Gly Ser Glu Lys Ala Arg Ala Ile 370 375 380Met Glu Glu Val Met Ser Leu Leu Gln Pro Leu Asn Ile Thr Gln Val385 390 395 400Leu Ser His Gly Glu Gly Thr Asp Ile Asn Phe Trp Ile Gln Ala Gly 405 410 415Val Pro Gly Ala Ser Leu Leu Asp Asp Leu Tyr Lys Tyr Phe Phe Phe 420 425 430His His Ser His Gly Asp Thr Met Thr Val Met Asp Pro Ser Arg Trp 435 440 445Met Leu Leu Leu Leu Phe Gly Leu Leu Phe Leu Met Leu Leu Gln Thr 450 455 460Trp Lys Lys Cys Cys Leu Gly Pro Arg Asn Ser Lys Lys Glu Thr Phe465 470 475 480Ser Cys Phe Trp Pro Gly Ile Leu Gly Leu Gln Leu Trp Lys Thr Pro 485 490 495Leu His Ile Thr Ile Ser Ser Asn Ser Ser Ser Lys His Asn Ser Ile 500 505 510Ser Cys Phe Leu Leu Leu Ser Phe Leu Ile Leu Ser Lys Phe Ser Asp 515 520 525Ser Arg Lys Arg Asn His Ser Pro Leu Pro Pro Thr Thr 530 535 54012441PRTHomo sapiens 12Met Val Pro Pro Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu1 5 10 15Ala Val Val Tyr Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Ala His 20 25 30Met Lys Ser Ser Ala Trp Val Asn Lys Ile Gln Val Leu Met Ala Ala 35 40 45Ala Ser Phe Gly Gln Thr Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser 50 55 60Val Gly Cys Thr Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe65 70 75 80Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu 85 90 95Trp Ile Pro Asn Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly 100 105 110Thr His Trp Leu Met Gly Asn Ile Leu Arg Leu Leu Phe Gly Ser Met 115 120 125Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr 130 135 140Pro Leu Val Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr145 150 155 160Ser Ala Ile Gly Ile Asp Leu Ala Ser His Gly Phe Ile Val Ala Ala 165 170 175Val Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp 180 185 190Gln Ser Ala Ala Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr 195 200 205Leu Lys Gln Glu Glu Glu Thr His Ile Arg Asn Glu Gln Val Arg Gln 210 215 220Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp225 230 235 240His Gly Lys Pro Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu 245 250 255Gln Leu Lys Asp Ser Ile Asp Arg Glu Lys Ile Ala Val Ile Gly His 260 265 270Ser Phe Gly Gly Ala Thr Val Ile Gln Thr Leu Ser Glu Asp Gln Arg 275 280 285Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp 290 295 300Glu Val Tyr Ser Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu305 310 315 320Tyr Phe Gln Tyr Pro Ala Asn Ile Ile Lys Met Lys Lys Cys Tyr Ser 325 330 335Pro Asp Lys Glu Arg Lys Met Ile Thr Ile Arg Gly Ser Val His Gln 340 345 350Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly His Met 355 360 365Leu Lys Leu Lys Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Leu Ser 370 375 380Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys His Leu Gly Leu His Lys385 390 395 400Asp Phe Asp Gln Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu 405 410 415Ile Pro Gly Thr Asn Ile Asn Thr Thr Asn Gln His Ile Met Leu Gln 420 425 430Asn Ser Ser Gly Ile Glu Lys Tyr Asn 435 44013212PRTHomo sapiens 13Met Met Gly Ile Pro Ile Arg Lys Phe Ile Cys Ala Ser Asn Gln Asn1 5 10 15His Val Leu Thr Asp Phe Ile Lys Thr Gly His Tyr Asp Leu Arg Glu 20 25 30Arg Lys Leu Ala Gln Thr Phe Ser Pro Ser Ile Asp Ile Leu Lys Ser 35 40 45Ser Asn Leu Glu Arg His Leu His Leu Met Ala Asn Asn Arg Leu Glu 50 55 60Ser Gln His His Phe Gln Ile Glu Lys Ala Leu Val Glu Lys Leu Gln65 70 75 80Gln Asp Phe Val Ala Asp Trp Cys Ser Glu Gly Glu Cys Leu Ala Ala 85 90 95Ile Asn Ser Thr Tyr Asn Thr Ser Gly Tyr Ile Leu Asp Pro His Thr 100 105 110Ala Val Ala Lys Val Val Ala Asp Arg Val Gln Asp Lys Thr Cys Pro 115 120 125Val Ile Ile Ser Ser Thr Ala His Tyr Ser Lys Phe Ala Pro Ala Ile 130 135 140Met Gln Ala Leu Lys Ile Lys Glu Ile Asn Glu Thr Ser Ser Ser Gln145 150 155 160Leu Tyr Leu Leu Gly Ser Tyr Asn Ala Leu Pro Pro Leu His Glu Ala 165 170 175Leu Leu Glu Arg Thr Lys Gln Gln Glu Lys Met Glu Tyr Gln Val Cys 180 185 190Ala Ala Asp Met Asn Val Leu Lys Ser His Val Glu Gln Leu Val Gln 195 200 205Asn Gln Phe Ile 2101414PRTHomo sapiens 14Cys Ser Asp Thr Ser Pro Asp Thr Glu Glu Ser Tyr Ser Asp1 5 101515PRTHomo sapiens 15Cys Ser Asp Leu Lys Arg Ser Leu Gly Phe Val Ser Lys Val Glu1 5 10 15169PRTHomo sapiens 16Cys Ala Glu Glu Glu Lys Glu Glu Leu1 5

Claims

1. A method of detecting the presence of HCC in a mammal comprising: a) obtaining a biological sample from the mammal; b) assaying the sample to quantify at least a non-cell-associated HCC related protein; and c) comparing the quantity of the non-cell-associated HCC related protein to a control level.

2. The method of claim 1 wherein assaying the sample is selected from the group consisting of using an enzyme linked immunosorbent assay (ELISA) and competition assays using monoclonal, polyclonal, or a combination of monoclonal and polyclonal antibodies.

3. The method of claim 1, wherein assaying the sample includes using a receptor molecule that interacts specifically with the non-cell-associated HCC related protein.

4. The method of claim 1, wherein assaying the sample includes an activity assay and the non-cell-associated HCC related protein is selected from the group consisting of an enzyme and involved in a quantifiable chemical or biological reaction.

5. The method of claim 2 wherein the polyclonal antibodies include those that bind PLA2G13.

6. A method of detecting the presence of HCC in a mammal comprising: a) obtaining a tissue sample from the mammal; b) assaying the sample to quantify at least one of a cell-associated HCC related protein; and c) comparing the quantity of cell-associated HCC related proteins to a control level.

7. The method of claim 6, wherein the tissue sample is obtained by biopsy.

8. The method of claim 6, wherein the tissue sample is a liver tissue sample.

9. The method of claim 6 wherein assaying the sample is selected from the group consisting of competition assays using monoclonal, polyclonal, or a combination of monoclonal and polyclonal antibodies.

10. The method of claim 9 wherein the polyclonal antibodies include those that bind PLA2G13.

11. A method of detecting HCC in a mammal comprising: a) injecting the mammal with a conjugate including a targeting reagent and an imaging agent; b) imaging the mammal; and c) evaluating the resulting image for the presence of at least one of a cell-associated HCC related protein.

12. The method of claim 11, wherein the targeting agent is selected from at least one of the group consisting of an antibody, a receptor and a ligand and wherein the antibody, receptor and ligand specifically interacts with at least one of the cell-associated HCC related proteins.

13. The method of claim 11 wherein the targeting agent is anti-PLA2G13.

14. The method of claim 11, wherein the imaging agent is selected from the group consisting of a dye, radioisotope and a compound that enhances the sensitivity of a scanning methodology selected from the group consisting of magnetic resonance imaging (MRI), ultrasound, computer assisted tomography (CT), single photon emission computer assisted tomography (SPECT) and immunoscintography.

15. (canceled)

16. A method of detecting HCC in a mammal comprising: a) obtaining a sample of the mammal's liver tissue; and b) employing at least one of the group consisting of an immunocytochemistry technique using a cell-associated HCC related protein-specific antibody conjugated to at least one imaging agent and an immunohistochemistry technique using a cell-associated HCC related protein-specific antibody conjugated to at least one imaging agent.

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