WO2005095455A1 - Antibody specific for parafibromin, a new marker of parathyroid carcinoma - Google Patents

Abstract

Expression of the HRPT2 gene, which encodes parafibromin, is reduced in parathyroid carcinoma as well as in other carcinomas. Antibodies specific for parafibromin are disclosed, along with their use to evaluate levels of parafibromin in tissue samples. Loss of immunoreactivity is a highly selective marker for parathyroid carcinoma, and is a useful marker for kidney and bladder cancer, as well as other carcinomas, and provides the first laboratory test that can serve as a standard for pathological diagnosis of parathyroid cancer.

Description

ANTIBODY SPECIFIC FOR PARAFIBROMIN, A NEW MARKER OF PARATHYROID CARCINOMA

BACKGROUND OF THE INVENTION Field of the Invention The present invention in the fields of molecular biology, immunology and medicine is directed to antibodies specific for a protein, parafibromin, the product of the HRPT2 gene. Suppression of the expression of this protein is a highly selective marker for parathyroid carcinoma. Such antibodies aι-e useful in diagnosis of this disease. Description of the Background Art The HRPT2 gene is a recently identified putative tumor suppressor gene, encoding a protein product named parafibromin (Howell VM et al, JMed Genet 2O03. 40:657-63; Shattuck TM et al., N Engl JMed 2003. 349: 1722-29). Somatic HRPT2 gene mutations are present in up to 100% of sporadic parathyroid carcinomas, and balladic inactivation is present in the majority of these carcinomas. Tumors with HRPT2 gene mutations have a distinct microarray genetic signature, suggesting the existence of novel parafibromin-mediated tumorigenesis pathways (Haven CJ et al., Proc Natl Acad Sci USA. (in press). Primary hyperparathyroidism is a common disorder that is diagnosed with increasing frequency (Grimelius, L et al, Semin Surg Oncol, 1997. 13:142-54). Between <1% and 5% of cases of primary hyperparathyroidism are due to parathyroid carcinoma (Shane, E., J Clin Endocrinol Metab, 2001. 86:485-93) with especially high rates reported in Japan (Obara, T et al Semin Surg Oncol, 1997. 13:134-41) and Italy (Favia, G et al, World J Surg, 1998, 22: 1225-30). The causes of this variation are unclear, but may reflect a true geographical difference, referral ^"bias or differences in histologic criteria used. There is no definitive standard for pathologic diagnosis of parathyroid carcinoma apart from the presence of local invasiveness or metastasis (Haven CJ et al, J Paihol 2004 202:86-94; Shane, supra) which diagnosis includes subjective elements. As a result of the limited state of the art in diagnostic ability, 86% of diagnosed parathyroid carcinomas may go unrecognized whether intraoperatively or even after histopathologic evaluation, which may result in inadequate surgical resection. Carcinoma is often only recognized retrospectively upon relapse, by which time treatment options are limited. In one series, half of all recurrent or metastatic carcinomas were initially diagnosed as benign (Sandelin, K. et al, World J Surg, 1994. 18:594-8; discussion 599) . Indeed, it has been concluded by some authors that systematic diagnosis of parathyroid carcinoma may rest on ongoing postoperative follow-up of patients who have undergone resection of apparently benign adenomas (Hara, H. et al, Diagn Cytopathol, 1998. 18:192-8; Wells, SA, Jr., J Bone Miner Res, 2002. 17 Suppl 2:N158-62). With recent evidence that patients with an apparently sporadic carcinoma may actually be manifesting a forme fruste of hyperparathyroidism-jaw tumor (HPT-JT) syndrome in about 20% of cases (Shattuck, T.M. et al. , N Engl J Med, 2003. 349 : 1722-9), a hereditary multi-tumor syndrome characterized by HRPT2 gene mutations (Jackson CE et al. (1990) Surgery 108:1006-12), the need for accuracy in diagnosis is even more critical. A correct diagnosis would allow timely definitive surgery and targeted genetic screening. From a clinical viewpoint, it is unsatisfactory to rely on tumor extension for diagnosis, as early recognition and treatment by definitive resection are the main determinants of prognosis Shane, supra; Obara. et al, supra; Hundahl, SA et al, 1999. 86:538-44; Clayman, GL et al, Cancer, 2004. 100:900-5) Thirty years ago, Schantz and Castleman (Cancer, 1973. 3i:600-5) first defined histopathologic guidelines for the diagnosis of carcinoma: uniform sheets of cells arranged in a lobulated fashion separated by fibrous trabeculae; capsular or vascular invasion; and the presence of mitotic figures. Unfortunately, mitotic features, fibrous bands and uniform sheets of cells were found not to be pathognomonic for parathyroid carcinoma (Haven, CJ et al, J Pathol, 2004-. 202:86-94; Clayman et al, supra) occurring in benign parathyroid adenomas so frequently as to limit their specificity. The difficulty of histopathology of the parathyroid is such that even the distinction on a microscopic level between adenoma and hyperplasia is unclear (Grimelius et al, supra), current criteria relying on the number of involved glands. As a result of the limited applicability of these guidelines resulting in subjectivity in carcinoma diagnosis, many adjunct investigations for parathyroid carcinoma have been studied, including electron microscopy, immunohistochemistry, DNA flow cytometry and in-situ hybridization (Shane et al, supra). Immunohistochemical markers that have been studied include retinoblastoma tumor suppressor gene protein (pRb), calcium-sensing receptor CASK., Ki-67, cytokeratin-14, p27, mdm2, Bcl-2, cyclin Dl, p53 and p21 (Haven et al, supra ., J Pathol, 2004. 202:86-94; Naccarato, AG et al, J Endocrinol Invest, 1998. 21:136-41; Stojadinovic, A et al, Hum Pathol, 2003. 34:54-64; Erickson, LA et al, Am J Surg Pathol, 2002. 26:344-9; Erickson, LA et al, Am J Surg Pathol, 1999. 23:288-95; Farnebo, F et al, World J Surg, 1999. 23:68-74; Lloyd, RV et al, Endocr Pathol, 1995. 6:279-287; Lloyd, RV et al, Endocr Pathol, 1995. 6:279-287; Vargas, MP et al, Mod Pathol, 1997. 10:12-17). However, many markers have not been demonstrated to be useful in this regard (Stojadinovic et al, supra) The most extensively studied marker is the retinoblastoma tumor suppressor gene KB and the KB protein (pRb). Cryns et al demonstrated allelic loss in 9 out of 9 specimens of parathyroid carcinoma, and 1 out of 19 specimens of parathyroid adenoma, using Southern blot methods (Cryns, VL et al, N Engl J Med, 1994. 330:757-61). In all studies since then, all but two carcinoma specimens have been reported as having loss of heterozygosity at 13q (Cetani, F et al, Gin Endocrinol (Oxf), 20O4. 60:99- 106; Yoshimoto, K et al, Clin Endocrinol (Oxf), 1998. 48:67-72) . However, several recent studies using more sensitive PCR-based methods have found that up to 29% of adenomas exhibit loss of heterozygosity as well (Tahara, H et al, Cane Res, 1996. 56:599-605). Thus, loss of heterozygosity of the KB gene is not specific to parathyroid carcinoma. Recently, mutation analysis of the KB gene did not show any mutations in 6 out of 6 parathyroid carcinomas (Shattuck, TM et al, Clin Endocrinol (Oxf), 2003. 59:180-9). Studies of pRb immunostaining have also yielded conflicting results. Some studies have reported that it may be a helpful marker (Cryns et al, supra; Cetani et al, supra), but several other studies have contradicted these findings (Farnebo et al, supra ; Loyd et al, supra). A recent review of KB gene abnormalities in parathyroid carcinoma concluded that no definitive conclusion comld be drawn with regards to pRb staining (Cetani et al, supra). Recent interest has focused on the use of cell cycle associated antigens. Erickson et ύ (1999, supra) noted that the mean p27 labelling index (percentage of positive nuclei) was lower in carcinoma in comparison to benign tissue. However, overlap between the two groups was also noted. Usi g a threshold of 30% stained tumor nuclei, Stojadinovic et al (supra) subsequently reported that 2/9 (22%) of carcinomas stained positive for p27, while 20% (9/36) of carcinomas stained negative. In. the same study, a multiple-marker phenotype including p27, Bcl-2, Ki-67 and mdm2 was useful in defining a subset of benign tumors, but carcinoma displayed a complex range of multi-marker phenotypes, some of which were not specific. DNA cytometry is of prognostic but not diagnostic use. It is useful for distinguishing a subset of parathyroid carcinomas which are aneuploid, and which may behave in a more aggressive fa.shion (Levin, KE et al, J Clin Endocrinol Metab, 1988. 67:779-84; Bondeson, L et al, Am J Surg Pathol, 1993. 17:820-9; August, DA et al, Surgery, 1993. 113:290-6). However, aneuploidy is not a_. useful indicator for diagnosing carcinoma, as aneuploidy also occurs frequently in benign tissue (Sϊiane, supra; Obara, supra; Obara, T et al., Cancer, 1990. 66:1555-62; Sandelin, supra) and about half of all parathyroid carcinomas do not display aneuploidy (Shattuck et al, supra; Levin et al, suprac; Bondeson et al, supra; August et al, supra ; Sandelin, K. World J Surg, 1992. 16:724-31. A correct diagnosis would allow early definitive surgery and targeted genetic screening of individuals and their families. A reliable standard would also clarify the true incidence of parathyroid carcinoma, which has ranged in various published case series from <1% to 5.2% of cases of primary hyperparathyroidism (Shane, supra). Hereditary Hyperparathyroidism Hereditary forms of hyperparathyroidism have been known for years. A large number of recent genetic studies on familial hypeφarathyroidism have resulted in a better understanding of their etiology and pathogenesis. Based on both clinical and molecular characterization, this heterogeneous group of diseases can be further divided into several subgroups such as MEN1, MEN2, familial benign hypocalciuric hypercalcemia and hyperparathyroidism-jaw tumor syndrome (HPT-JTS) (Villablanca A et al. (2001) J Endocrinol Genet 2:3-12). In addition, it is important to appreciate that some of these syndromes have an allelic or "milder" counteφart in the form of familial isolated hypeφarathyroidism - hypeφarathyroidism without any other features or endocrinopathies - which results from mutations of the same gene. Hyperparathyroidism-Jaw Tumor Syndrome (HPT-JTS) HPT-JTS is an autosomal dominant disorder characterized by parathyroid adenoma or carcinoma, fibroosseous lesions (ossifying fibroma) of the mandible and maxilla, and renal cysts and tumors. Since it is a relatively recently described entity, its incidence or prevalence is unknown. About 80% of patients present with hypeφarathyroidism (Caφten JD et al. (2002) Nature Genet 32:676-80), that may develop in late adolescence or beyond. There is reduced penetrance in females (Teh BT et al. (1996) J Clin Endocrinol Metab 57:4204-4211). Parathyroid carcinoma occurs in approximately 10-15 % of affected individuals (Caφten et al, supra). Compared with MEN 1 -related hypeφarathyroidism, HTP-JTS appears to run a more aggressive course with more severe hypercalcemia. The incidence of parathyroid carcinoma is also much higher than in other endocrine related disorders. Primary hypeφarathyroidism in HPT-JTS more often involves ne or two glands (adenoma or double adenoma) that may or may not present synchronously (Jackson CE et al. (1990) Surgery 705:1006-12). In contrast, in MEN1, all four glands are frequently involved. The other unique feature of parathyroid neoplasia in HPT-JTS is the high incidence of cystic changes. The combination of recurring adenomas often with cystic change has prompted some to refer to this entity as "familial cystic parathyroid adenomatosis" (Mallette LE et al. (1987) Ann Intern Med 107:54-60) without regard to the associated jaw lesions. In many reports of parathyroid carcinoma associated with HPT-JTS (Caφten et al, supra; Dinnen et al, supra; Cavaco BM et al. (2001) QJM 94:213-22; Szabo J, et al. (1995) Am JHum Genet 56:944-50; Kakinuma A et al. (1994) Intern Med 33:123-6; Rosen IB et al (1981) Am JSurg 142:494- 8), the diagnosis of carcinoma was based upon several features including fibrous bands, recurrence in the area of previous surgery, possible capsular or perineural invasion, invasion of adjacent thyroid parenchyma or distant metastasis. As noted above, histological diagnosis is extremely difficult. However, thick fibrous bands are not restricted to parathyroid carcinoma and are also observed in longstanding parathyroid disease, often associated with cystic change. Jaw Tumors The term "jaw tumor" is actually a misnomer, as it is now known that patients may develop the tumors not only in the jaw (mandible) but also in the maxilla. These tumors are less well defined than cystic parathyroid adenomatosis in HPT-JTS. The classical bone lesion of hypeφarathyroidism, osteitis fibrosa cystica, consists of a relatively vascular fibroblast-rich stroma with irregularly distributed clusters of osteoclast-type giant cells. These lesions tend to resolve slowly (months to years) after correction of the hypeφarathyroidism, (Sciubba JJ et al. (2001) Armed Forces Institute of Pathology 1- 275). In contrast, a subset of jaw lesions associated with familial hypeφarathyroidism do not resolve with correction of the hypeφarathyroidism (Teh et al (1996) supra; Jackson et al, supra; Mallette et al, supra; Kennett et al, supra; Dinnen et al, supra; Fujikawa et al, supra; Cavaco et al, supra; Szabo et al, supra; Kakinuma et al, supra; Rosen et al, supra; Haven CJ et al. (2000) J Clin Endocrinol Metab 85: 1449-54; Inoue H et al. (1995) Clin Endocrinol (Oxf) 43 :225-9; Warnakulasuriya S et al. (1985)

Oral Surg Oral Med Oral Pathol 59:269-74; Jackson CE (1958) Ann Intern Med 49:^,29-36; Firat D et al. (1985) Am JMed 44:421-9). These bone lesions have also been found in HPT-JTS patients who have no evidence of hypeφarathyroidism. At least 13 reports describe one or more kindreds consistent with HPT-JTS in which at least one family member underwent surgical resection of the jaw tumor and a specific histological diagnosis was reported. Most of these reports described the jaw lesion as histologically distinct from the typical bone lesions expected with hypeφarathyroidism. While these jaw lesions are obviously distinct from osteitis fibrosa cystica there has been some debate as to their classification as fibrous dysplasia or ossifying fibroma. The more recent reports refer to these lesions as ossifying fibroma or variants of ossifying fibroma (cementifying fibroma, cemento- ossifying fibroma) , but at least one earlier report (Wamalculasuriya S et al. (1985) Oral Surg Oral Med Oral Pathol 59:269-14) clearly illustrated histologically what appears to be the lesional bone fusing with adjacent uninvolved cancelous and cortical bone. Until more cases of the jaw lesions of HPT-JTS have been described and the histopathologic features of fibrous dysplasia, ossifying fibroma, and variants of each of these have been clarified, it may be prudent to refer to the former cases as merely fibroosseous lesions. Such nomenclature effectively distinguishes these cases from the most common bone lesion of hypeφarathyroidism — osteitis cystica fibrosa. Kidney Lesions In addition to cystic adenomatosis and jaw lesions, a wide spectrum of other tumors has been associated with HPT-JTS, most notably renal lesions (Teh et al, (1996) supra; Szabo et al, supra; Kakinuma et al, supra. In the two families reported by Teh et al. (1996), 5 individuals in one kindred and 2 in the other had renal lesions. In the latter kindred, polycystic kidney disease was predominant, while in the other kindred, in addition to renal cysts, several adults had large cystic tumors comprising mesenchymal and epithelial elements. Another renal tumor, Wilm's tumor, has been reported with HPT- JTS is, (Szabo et al, supra; Kakinuma et al, supra) in three separate individuals from separate families. Prognosis The majority of patients with adenoma are cured by surgery; recurrence is not as common as in MEN1 patients. However, prognosis is guarded once parathyroid carcinoma is confirmed (see above). In all the reported cases, the jaw lesions involved the premolar or molar areas of the mandible or maxilla. While most of these lesions involve either the mandible or the maxilla on one side, bilateral or multifocal lesions have also been described (Cavaco et al, supra; Wamakulasuriya et al, supra; Firat et al, supra). While none of the described jaw tumors have been malignant, some have recurred (although it is difficult to distinguish incomplete initial resection from de novo lesions. There is thus a need in the art for novel approaches that permit earlier diagnosis of parathyroid carcinoma and the distinction of this disease category from other syndromes that share characteristics. The present invention is the first to provide a rather simple and economical solution to this need. Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents. SUMMARY OF THE INVENTION Despite many studies over the years, diagnosis of parathyroid carcinoma has remained a major challenge for expert pathologists. Based on the evidence of a high prevalence of HRPT2 gene mutations in parathyroid carcinoma, the present inventors conceived that loss of parafibromin immunoreactivity permits one to distinguish parathyroid carcinomas from benign tissue, including adenomas, hypeφlasias and MEN1 -associated tumors. The present inventors have produced a novel mAb specific for parafibromin and discovered a very strong association between loss of parafibromin immunoreactivity in a tissue sample and a diagnosis of parathyroid carcinoma. Subsequently, this association was also found for other carcinomas, most notably, bladder and kidney (clear cell-renal cell carcinoma or "CC-RCC"). Similar findings are expected with lung, breast, prostate and colorectal carcinomas, wherein loss of parafibromin reactivity is indicative of cells/tissues being carcinomatous, or is predictive of a poorer prognosis. The present invention provides antibody specific for an epitope of human parafibromin. The expression of this epitope (and of parafibromin) is reduced in or on a carcinoma cell or tissue when compared to normal corresponding tissue. This has been demonstrated for bladder, kidney and most definitively for parathyroid carcinoma. Preferably, the antibody is specific for an epitope formed by or created by the sequence

RRPDRKDLLGYLN (SEQ ID NO:3), or an epitope comprising this sequence. Preferably, the antibody is a monoclonal antibody (mAb), and it may be a chimeric, humanized or human mAb. A preferred mAb is murine mAb 2H1 exemplified herein and deposited in the ATCC under accession number , which is an IgGl ,κ, or another mAb that binds specifically to the same parafibromin epitope does 2H1. Other antibody compositions provided herein include any antibody as above that is immobilized to a solid support. The antibody may be labeled with a detectable label. Another embodiment is a diagnostically useful antibody composition comprising (a) the above antibody labeled with a diagnostically useful label; and (b) a diagnostically acceptable carrier. Detectable labels include a radionuclide, a PET-imageable agent, an MRI-imageable agent, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer. Preferred radionuclides include ³H, ¹⁴C, ³⁵S, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁷Ru, ⁹⁹Tc, ^ιπIn, ^,23I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb and ²⁰¹Tl. The label may be a fluorescer or fluorogen selected from the group consisting of fluorescein, rhodamine, dansyl, phycoerythrin, phycocyamn, allophycocyanin, o-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green and Texas Red. The present invention provides a method for producing a hybridoma cell line that produces a mAb specific for an epitope of human parafibromin, preferably an epitope foπned by or comprising RRPDRKDLLGYLN (SEQ ID NO:3), the method comprising: (a) immunizing a donor animal with a parafibromin peptide or polypeptide conjugated to a carrier, a preferred immunogen being RRPDRKDLLGYLNC (SEQ ID NO:4) conjugated to a preferred carrier such as keyhole limpet hemocyanin (KLH); (b) obtaining B lymphocytes from the immunized donor animal;

(c) fusing the B lymphocytes with cells of a fusion partner cell line to obtain hybrid cells;

(d) selecting hybrid cells which produce the parafibromin-specific antibody by: (i) culturing the hybrid cells; (ii) screening the medium of the cultures for the presence of an antibody which binds to parafibromin in an immunoassay or to cells expressing parafibromin, preferably in an immunohistochemical assay, thereby detecting antibody producing hybrid cells, and (e) growing the selected hybrid cells, thereby producing the hybridoma cell line. Also provided is a method for producing the above a mAb, comprising

(a) culturing a hybridoma cell line produced as above, or produced by a different method, under conditions which permit antibody production and secretion by the cell line;

(b) obtaining the culture medium containing the antibody; and

(c) optionally, purifying the antibody. The present invention is also directed to an immunoassay method for detecting a parathyroid carcinoma in a test sample, preferably a tissue sample, comprising:

(a) contacting the test sample with an antibody or diagnostic composition, as above, and

(b) measuring the amount of antibody bound to the test sample compared to a control sample, preferably as tissue staining, wherein a loss of parafibromin immunoreactivity in the test sample compared to the control is indicative of any carcinoma, as tested specifically for bladder, kidney and most notably, parathyroid carcinoma. The staining in the above method may be produced by a detectably labeled ligand for the antibody, such as an anti-immunoglobulin "secondary" antibody. Also provided is an imaging method for detecting carcinoma, preferably parathyroid carcinoma, in a subject, comprising:

(a) administering to the subject the above antibody or composition wherein the antibody is labeled with an imageable label; and

(b) measuring by an imaging technique the accumulation of the antibody in a tissue or region in which the carcinoma is suspected, and comparing the accumulation to the antibody accumulation in unaffected comparable tissue, such as normal parathyroid, wherein a decrease in antibody accumulation the suspected tissue or region compared to the unaffected tissue is indicative of the presence of carcinoma. Preferred imaging techniques are MRI and PET scanning. The invention includes a kit useful in a method of measuring or detecting parafibromin immunoreactivity in a sample, the kit being adapted to receive therein one or more containers, the kit comprising:

(a) the above antibody in detectably labeled form or the above diagnostic composition; and

(b) optionally, one or more additional reagents useful for carrying out an immunoassay. In the above kit the antibody is directly labeled with a radionuclide, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer. In another kit embodiment, the kit comprises: (a) as a first antibody, the anti-parafibromin antibody of the invention, and (b) one or more additional reagents useful for carrying out an immunoassay, which include a detectably labeled ligand for the first antibody. A preferred ligand is a labeled second antibody specific for the first antibody, i.e., anti-Ig.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides confocal images demonstrating co-localization of the green fluorescent protein (GFP)-parafibromin fusion protein with anti-parafibromin antibody within the nuclei of transfected HEK293 cells. From left: GFP-parafibromin fusion protein expression (green); Anti- parafibromin mAb binding, as detected by secondary Rhodamine-Red goat anti-mouse antibody (red); Nomarski image of cells; DAPI staining of nuclei (blue); Superimposition of all images demonstrated co-localization within nuclei. All images were captured with a Zeiss LSM510 META laser-scanning confocal microscope Figure 2 is a Western blot showing an increase in parafibromin expression in COS7 cells transfected with pcDNA3-HRPT2, compared to cells transfected with empty vector, as detected by anti- parafibromin antibody. The control was β-actin. Figure 3A-3H show results of immunohistochemical staining representing the various staining patterns manifested in the different pathologies through parafibromin immunostaining. Figs 3A- 3D are at a magnification of 200x. Fig. 3 A shows diffuse staining of primary parathyroid hypeφlasia); Fig. 3B shows diffuse staining of sporadic adenoma with a rim of normal tissue. Figs 3C and 3D show, respectively, focal loss and diffuse loss (associated with parathyroid carcinoma). Figs. 3E- 3H are higher magnifications (400x) of the respective parathyroid pathologies. All images were taken with a Spot Insight Camera on a Nikon Eclipse E600.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present inventors have discovered that a loss or fall of parafibromin expression is associated with parathyroid carcinoma and have produced a mAb to parafibromin and developed methods for detecting/diagnosing parathyroid carcinoma in a subject by examining tissue samples using this mAb. The present invention is further directed to other diagnostic methods and compositions based on the foregoing discovery. The terms "parafibromin" and HRPT2 (when referring to the protein product of this gene) are used interchangeably herein.

Human HRPT2 sequences The nucleic acid sequence encoding human HRPT2 which encodes a protein given the name "parafibromin" is shown below (SEQ ED NO:l)

1 gctactgccc ctgctgctgt cgtaggcgag gacggctgtt agtgctgctg ctgttggttc

61 gtcgcggcgg cgaaggagga ggaggaagag ggcgaggcga caagagaaga aggaggcagg

121 cgcggcggca gcggcggcgc cccgagccgg cggaggcgag gggggggaag atggcggacg

181 tgcttagcgt cctgcgacag tacaacatcc agaagaagga gattgtggtg aagggagacg

241 aagtgatctt cggggagttc tcctggccca agaatgtgaa gaccaactat gttgtttggg

301 ggactggaaa ggaaggccaa cccagagagt actacacatt ggattccatt ttatttctac

361 ttaataacgt gcacctttct catcctgttt atgtccgacg tgcagctact gaaaatattc

421 ctgtggttag aagacctgat cgaaaagatc tacttggata tctcaatggt gaagcgtcaa

481 catcggcaag tatagacaga agcgctccct tagaaatagg tcttcagcga tctactcaag

541 tcaaacgagc tgcagatgaa gttttagcag aagcaaagaa accacgaatt gaggatgaag

601 agtgtgtgcg ccttgataaa gagagattgg ctgcccgttt ggagggtcac aaagaaggga

661 ttgtacagac tgaacagatt aggtctttgt ctgaagctat gtcagtggaa aaaattgctg

721 caatcaaagc caaaattatg gctaagaaaa gatctactat caagactgat ctagatgatg

781 acataactgc ccttaaacag aggagttttg tggatgctga ggtagatgtg acccgagata

841 ttgtcagcag agagagagta tggaggacac gaacaactat cttacaaagc acaggaaaga

901 atttttccaa gaacattttt gcaattcttc aatctgtaaa agccagagaa gaagggcgtg

961 cacctgaaca gcgacctgcc ccaaatgcag cacctgtgga tcccactttg cgcaccaaac

1021 agcctatccc agctgcctat aacagatacg atcaggaaag attcaaagga aaagaagaaa

1081 cggaaggctt caaaattgac actatgggaa cctaccatgg tatgacactg aaatctgtaa

1141 cggagggtgc atctgcccgg aagactcaga ctcctgcagc ccagccagta ccaagaccag

1201 tttctcaagc aagacctccc ccaaatcaga agaaaggatc tcgaacaccc attatcataa

1261 ttcctgcagc taccacctct ttaataacca tgcttaatgc aaaagacctt ctacaggacc

1321 tgaaatttgt cccatcagat gaaaagaaga aacaaggttg tcaacgagaa aatgaaactc

1381 taatacaaag aagaaaagac cagatgcaac cagggggcac tgcaattagt gttacagtac

1441 cttatagagt agtagaccag ccccttaaac ttatgcctca agactgggac cgcgttgtag

1501 ccgtttttgt gcagggtcct gcatggcagt tcaaaggttg gccatggctt ttgcctgatg

1561 gatcaccagt tgatatattt gctaaaatta aagccttcca tctgaagtat gatgaagttc

1621 gtctggatcc aaatgttcag aaatgggatg taacagtatt agaactcagc tatcacaaac

1681 gtcatttgga tagaccagtg ttcttacggt tttgggaaac attggacagg tacatggtaa

1741 agcataaatc gcacttgaga ttctgaatta tttggctcct ccatttctgg aaattgagac

1801 tcaagcttta tgaatttatc aagaacttaa aaatgaagaa ggtcacagat tgatctttta

1861 taagacctta tttgatgctt tgtgcttcaa ggagatgata cctgtcatcc atataagcaa

1921 actttttggc ttacaactat ttttttaata ttagccttct agtctgtaat ggaaattgta

1981 tattttgata gaagtttttt ctccattggt taaattagca ttacttaaaa tttgtttctt

2041 tagaaaataa atgcaggtta taaatgtgtg tatatttaga gattataagg ctctctgagc

2101 catcttctga tttttcattg ctctataatt ctttttactg aaaatactat gttatgaatg

2161 gtattaaatt ttagtctctg gaacatccaa aaccaagcaa agggatgtga ctattttgaa

2221 tgaatcagaa tgtcaacttg tatgtacact atatctacac ttactcatta tttaaaaaga

2281 ataatgaaaa atctagatca attcttcaat ttgattgaac tgttcagcct tttcaagatt

2341 tctttattta caaatgatta catttaaatg aatgtacatt cttctcactg actttggtga

2401 ttttgaaacc tagaatgatg tgtttctatc tgtaatatct ttccatttga aaaaaatctc

2461 aaaacacaga ttaaaaccac aataggctgt agtatttttt attttgggag ccagagtatg

2521 atttggggga agaatatgta tcagccctat tgcagtataa ctttaagctc cttttctctt

2581 tagtccactt ttgattgtaa tttttatggt ataggatttt gaatcttcta ttttaggctt

2641 gtcagtcttg gagttcttat cttccattat ccctaaatat tgataaactc ccaggcacca

2701 aagaaaacat ttgcttaatt gtctgaaaag aaacaagaga aaaacactgg tatttttatg

2761 tctgtattca atatggtata aaatataaaa actatatttt aacttagtga aatattttac

2821 tatttctcta cttcagacaa aatgttgcat ccaaggtaca tcaagtgacc atttgccttg

2881 aaccttgatt tcactttgtt tttttttttt ccttaaaggc aactaggaag ctttactttc

2941 ctaaagtgtt tttgccattg gaatttttgc tgatcacagt

The amino acid sequence of human parafibromin (SEQ ED NO: 2) encoded by the human HRPT2 gene coding sequence (SEQ ED NO:l) or a sequence variant of said coding sequence, is:

MADVLSVLRQ YNIQKKΞIW KGDEVIFGΞF S PKNVKTNY W GTGKEGQ PREYYTLDSI 60

LFLLNNVHLS HPVYVRRAAT ENIPWRRPD RKD LGY NG EASTSASIDR SAPLEIGLQR 120

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VTVPYRWDQ PLKLMPQDWD RWAVFVQGP A QFKGWP L LPDGSPVDIF AKIKAFH KY 480 DEVRLDPNVQ KWDVTVLE S YH RHLDRPV FLRFWETLDR YMVKHKSHLR F 531 The peptide fragment of SEQ ID NO:2 that the present inventors used to produce a mAb is RRPDRKDLLGYLN (SEQ ID NO:3). This peptide corresponds to residues 93-105 of parafibromin (based on the numbering of an aligned sequence) or residues 87-99 of SEQ ID NO:2, A preferred peptide, that was actually used as an immunogen by the present inventors, has a C- terminal Cys residue added to SEQ ID NO:3, resulting in RRPDRKDLLGYLNC (SEQ ID NO:4). Selection of this internal peptide (and artificial sequence SEQ ED NO:4) for use is based on Its high degree of homology to the homologues of HRPT2 in Drosophila and C. elegans. Second, the N-terminal 7 residues are hydrophilic and therefore more likely to be exposed on the surface of the folded native protein. The Cys residue was added at the C-terminus as a site for coupling of the peptide to a carrier protein, such as KLH.

ANTIBODIES SPECIFIC FOR EPITOPES OF PARAFIBROMIN In the following description, reference will be made to various methodologies known to those of skill in the art of immunology, cell biology, and molecular biology. Publications and other materials setting forth such known methodologies to which reference is made are incoφorated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of immunology include A.K. Abbas et al, Cellular and Molecular Immunology (Fourth Ed.), W.B. Saunders Co., Philadelphia, 2000; CA. Janeway et al, Immunobiology. The Immune System in Health and Disease, Fourth ed., Garland Publishing Co., New York, 1999; Roitt, I. et al, Immunology, (current ed.) C.V. Mosby Co., St. Louis, MO (1999); Klein, J., Immunology, Blackwell Scientific Publications, Inc., Cambridge, MA, (1990). Monoclonal antibodies (mAbs) and methods for their production and use are described in Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Hartlow, E. et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988); Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, NY (1980); H. Zola et al, in Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, 1982)). Immunoassay methods are also described in Coligan, J.E. et al, eds., Current Protocols in Immunology, Wiley-Interscience, New York 1991(or current edition); Butt, W.R. (ed.) Practical Immunoassay: Tlie State of the Art, Dekker, New York, 1984; Bizollon, Ch. A., ed., Monoclonal Antibodies and New Trends in Immunoassays, Elsevier, New York, 1984; Butler, J.E., ELISA (Chapter 29), In: van Oss, CJ. et al, (eds), IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J.E. (ed.), Immunochemistry of Solid-Phase Immunoassay , CRC Press, Boca Raton, 1991; Weintraub, B., Principles of Radioimmunoassays , Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986; Work, T.S. et al, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, (1978) ; Dabbs, DJ, Diagnostic Immunohistochemistiy, Churchill Livingstone, 2001. Anti-idiotypic antibodies are described, for example, in Idiotypy in Biology and Medicine,

Academic Press, New York, 1984; Immunological Reviews Volume 79, 1984; Immunological Reviews Volume 90, 1986; Curr. Top. Microbiol, Immunol. Volume 119, 1985; Bona, C. et al, CRC Crit. Rev. Immunol, pp. 33-81 (1981); Jerne, NK, Ann. Immunol 125Q.373-389 (1974); Jeme, NK, In: Idiotypes - Antigens on the Inside, Westen-Schnurr, I, ed., Editiones Roche, Basel, 1982, Urbain, J et al, Ann. Immunol. 133DΛ79- (1982); Rajewsky, K. et al, Ann. Rev. Immunol. 1:569-607 (1983) The present invention provides antibodies, both polyclonal and mAb, reactive with epitopes of parafibromin. The antibodies may be xenogeneic, allogeneic, syngeneic, or modified forms thereof, such as humanized or chimeric antibodies. Antiidiotypic antibodies specific for the idiotype of an anti- parafibromin antibody are also included. The term "antibody" is also meant to include both intact molecules as well as fragments thereof that include the antigen-binding site and are capable of binding to a parafibromin epitope. These include , Fab and F(ab')₂ fragments which lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl Med. 24:316-325 (1983)). Also included are Fv fragments (Hochman, J. et al. (1973) Biochemistry 72:1130-1135; Sharon, J. et al( 1976) Biochemistry 15:1591- 1594).). These various fragments are be produced using conventional techniques such as protease cleavage or chemical cleavage (see, e.g., Rousseaux et al, Meth. Enzymol, 121:663-69 (1986)) Polyclonal antibodies are obtained as sera from immunized animals such as rabbits, goats, rodents, etc. and may be used directly without further treatment or may be subjected to conventional enrichment or purification methods such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see Zola et al. , supra). An immunogen for generation of anti-parafibromin antibodies may comprise the complete parafibromin (HRPT2) protein, or an epitope-bearing fragments or derivative thereof. Useful immunogens are produced in a variety of ways known in the art, e.g., expression of cloned genes using conventional recombinant methods, isolation from cells of origin, cell populations expressing high levels of parafibromin, etc. In the case of shorter fragments, they are chemically synthesized. A preferred immunogen, as noted above and discussed below, is a carrier conjugated form of the parafibromin peptide RRPDRKDLLGYLNC (SEQ ID NO:4). The mAbs may be produced using conventional hybridoma technology, such as the procedures introduced by Kohler and Milstein (Nature, 256:495-97 (1975)),-and modifications thereof (see above references). An animal, preferably a mouse is primed by immunization with an immunogen as above to elicit the desired antibody response in the primed animal. B lymphocytes from the lymph nodes, spleens or peripheral blood of a primed, animal are fused with myeloma cells, generally in the presence of a fusion promoting agent such as polyethylene glycol (PEG). Any of a number of murine myeloma cell lines are available for such use: the P3-NSl/l-Ag4-l, P3-x63-k0Ag8.653, Sp2/0-Agl4, or HL1-653 myeloma lines (available from the ATCC, Rockville, MD). Subsequent steps include growth in selective medium so that unfused parental myeloma cells and donor lymphocyte cells eventually die while only the hybridoma cells survive. These are cloned and grown and their supernatants screened for the presence of antibody of the desired specificity, e.g., by immunoassay techniques using the parafibromin-Ig fusion protein Positive clones are subcloned, e.g., by limiting dilution, and the mAbs are isolated. Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art (see generally Fink et al, Prog. Clin. Pathol, 9: 121-33 (1984)). Generally, the individual cell line is propagated in culture and the culture medium containing high concentrations of a single mAb can be harvested by decantation, filtration, or centrifugation. human, such as a primate, or a human cell. For example, a B lymphocyte which produces anti-parafibromin antibody may be infected and transformed with a virus such as Epstein-Ban: vims to yield an immortal anti- parafibromin producing cell (Kozbor et al. Immun Today ^:72-79 (1983)). Alternatively, the B lymphocyte may be transformed by providing a transforming gene or transforming gene product, as is well-known in the art. Preferably, the antigen binding region will he of^" murine origin. In other embodiments, the antigen binding region may be derived from other animal species, in particular rodents such as rat or hamster. The human parafibromin-specific murine or chimeric mAb of the present invention may be produced in large quantities by injecting hybridoma or transfectoma cells secreting the antibody into the peritoneal cavity of mice and, after appropriate time, harvesting the ascites fluid which contains a high titer of the mAb, and isolating the mAb therefrom. For such in vivo production of the mAb with a non-murine hybridoma (e.g., rat or human), hybridoma cells are preferably grown in inadiated or athymic nude mice. Alternatively, the antibodies my be produced by culturing hybridoma (or transfectoma) cells in vitro and isolating secreted mAb from the cell culture medium. Human genes which encode the constant C regions of the chimeric antibodies of the present invention may be derived from a human fetal liver library or from any human cell including those which express and produce human Igs. The human C_H region can be derived from any of the known classes or isotypes of human H chains, including γ, μ, α, δ or ε, and subtypes thereof, such as Gl, G2, G3 and G4. Since the H chain isotype is responsible for the various effector functions of an antibody, the choice of C_H region will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity (ADCC). Preferably, the C_H region is derived from γl (IgGl), γ3 (IgG3), γ4 (IgG4), or μ (IgM). The human C region can be derived from either human L chain isotype, K or λ. Genes encoding human immunoglobulin C regions are obtained from human cells by standard cloning techniques (Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). Human C region genes are readily available from known clones containing genes representing the two classes of L chains, the five classes of H chains and subclasses thereof. Chimeric antibody fragments, such as F(ab')₂ and Fab, can be prepared by designing a chimeric H chain gene which is appropriately truncated. For example, a chimeric gene encoding an H chain portion of an F(ab')₂ fragment would include DNA sequences encoding the CHi domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule. Generally, the chimeric antibodies of the present invention are produced by cloning DNA segments encoding the H and L chain antigen-binding regions of a parafibromin-specific antibody, preferably non- human, and joining these DNA segments to DNA segments encoding human C_H and C_L regions, respectively, to produce chimeric immunoglobulin-encoding genes. Thus, in a prefened embodiment, a fused gene is created which comprises a first DNA segment that encodes at least the antigen-binding region of non-human origin, such as a functionally rearranged V region with joining (J) segment, linked to a second DNA segment encoding at least a part of a human C region. The DNA encoding the antibody-binding region may be genomic DNA or cDNA. A convenient alternative to the use of chromosomal gene fragments as the source of DNA encoding the murine V region antigen-binding segment is the use of cDNA for the construction of chimeric Ig genes, as reported by Liu et al. (Proc. Natl. Acad. Sci, USA 84:3439 (1987) and J. Immunology 139:3521 (1987), which references are hereby incoφorated by reference. The use of cDNA requires that gene expression elements appropriate for the host cell be combined with the gene in order to achieve synthesis of the desired protein. The use of cDNA sequences is advantageous over genomic sequences (which contain introns), in that cDNA sequences can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems. Therefore, in an embodiment utilizing cDNA encoding the antibody V region, the method of producing the chimeric antibody involves several steps, outlined below: 1. Isolation of messenger RNA (mRNA) from the cell line producing the mAb antibody, cloning and cDNA production therefrom; 2. Preparation of a full length cDNA library from puri fϊed mRNA from which the appropriate V region gene segments of the L and H chain genes can be: (i) identified with appropriate probes, (ii) sequenced, and (iii) made compatible with a C gene segment; 3. Preparation of C region gene segments by cDNA preparation and cloning;

4. Construction of complete H or L chain coding sequences by linkage of the cloned specific V region gene segments to cloned human C region gene, as described above;

5. Expression and production of chimeric L and H chains in selected hosts, including prokaryotic and eukaryotic cells. One common feature of all immunoglobulin H and L chain genes and their encoded mRNAs is the J region. H and L chain J regions have different sequences, but a high degree of sequence homology exists (greater than 80%) among each group, especially near the C region. This homology is exploited in this method and consensus sequences of H and L chain J regions may be used to design oligonucleotides for use as primers for introducing useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA vectors prepared from human cells can be modified by site-directed mutagenesis to place a restriction site at the analogous position in the human sequence. For example, one can clone the complete human kappa chain C ( ) region and the complete human γ-1 C region (Cγ-i). In this case, the alternative method based upon genomic C region clones as the source for C region vectors would not allow these genes to be expressed in bacterial systems where enzymes needed to remove intervening sequences are absent. Cloned V region segments are excised and ligated to L or H chain C region vectors. Alternatively, the human C_y_ι region can be modified by introducing a termination codon thereby generating a gene sequence which encodes the H chain portion of an Fab molecule. The coding sequences with linked V and C regions are then transferred into appropriate expression vehicles for expression in appropriate hosts, prokaryotic or eukaryotic. Two coding DNA sequences are said to be "operably linked" if the linkage results in a continuously translatable sequence without alteration or interruption of the triplet reading frame. A DNA coding sequence is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence. Expression vehicles include plasmids or other vectors. Preferred among these are vehicles carrying a functionally complete human C_H or C_L chain sequence having appropriate restriction sites engineered so that any V_H or V_L chain sequence with appropriate cohesive ends can be easily inserted therein. Human C_H or C_L chain sequence-containing vehicles thus serve as intermediates for the expression of any desired complete H or L chain in any appropriate host. A chimeric mouse-human antibody will typically be synthesized from genes driven by the chromosomal gene promoters native to the mouse H and L chain V regions used in the constructs; splicing usually occurs between the splice donor site in the mouse J region and the splice acceptor site preceding the human C region and also at the splice regions that occur within the human C_H region; polyadenylation and transcription termination occur at native chromosomal sites downstream of the human coding regions. Gene expression elements useful for the expression of cDNA genes include: (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama, H. et al, Mol. Cell. Biol. 3:280 (1983)), Rous sarcoma virus LTR (Gorman, C. et al, Proc. Natl. Acad. Sci, USA 79:6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl, R. et al, Cell 47:885 (1985)); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayama et al, supra); and (c) polyadenylation sites such as in SV40 (Okayama et al, supra). Immunoglobulin cDNA genes may be expressed as described by Liu et al, supra, and Weidle et al., Gene 57:21 (1987), using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit β-globin intervening sequence, Ig and rabbit β-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin genes comprised of part cDNA, part genomic DNA (Whittle et al, Protein Engineering 7:499 (1987)), the transcriptional promoter is human cytomegalovirus, the promoter enhancers are cytomegalovirus and mouse/human immunoglobulin, and mRNA splicing and polyadenylation regions are from the native chromosomal immunoglobulin sequences. In one embodiment, for expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are either or both the mouse Ig H chain enhancer and the viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence conesponding to the Ig chain being synthesized. In other embodiments, cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells. Each fused gene is assembled in, or inserted into, an expression vector. Recipient cells capable of expressing the chimeric immunoglobulin chain gene product are then transfected singly with a chimeric H or chimeric L chain-encoding gene, or are co-transfected with a chimeric H and a chimeric L chain gene. The transfected recipient cells are cultured under conditions that permit expression of the incoφorated genes and the expressed immunoglobulin chains or intact antibodies or fragments are recovered from the culture. In one embodiment, the fused genes encoding the chimeric H and L chains, or portions thereof, are assembled in separate expression vectors that are then used to co-transfect a recipient cell. Each vector may contain two selectable genes, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, wherein each vector has a different pair of genes. This strategy results in vectors which first direct the production, and permit amplification, of the fused genes in a bacterial system. The genes so produced and amplified in a bacterial host are subsequently used to co-transfect a eukaryotic cell, and allow selection of a co-transfected cell carrying the desired transfected genes. Examples of selectable genes for use in a bacterial system are the gene that confers resistance to ampicillin and the gene that confers resistance to chloramphenicol. Prefened selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyl transferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Selection of cells expressing gpt is based on the fact that the enzyme encoded by this gene utilizes xanthine as a substrate for purine nucleotide synthesis, whereas the analogous endogenous enzyme cannot. In a medium containing (1) mycophenolic acid, which blocks the conversion of inosine monophosphate to xanthine monophosphate, and (2) xanthine, only cells expressing the gpt gene can survive. The product of the neo blocks the inhibition of protein synthesis by the antibiotic G418 and other antibiotics of the neomycin class. The two selection procedures can be used simultaneously or sequentially to select for the expression of immunoglobulin chain genes introduced on two different DNA vectors into a eukaryotic cell. It is not necessary to include different selectable markers for eukaryotic cells; an H and an L chain vector, each containing the same selectable marker can be co-transfected. After selection of the appropriately resistant cells, the majority of the clones will contain integrated copies of both H and L chain vectors. Alternatively, the fused genes encoding the chimeric H and L chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the chimeric antibody, the preferred recipient cell line is a myeloma cell. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. A particularly preferred recipient cell is the Ig-non-producing myeloma cell SP2/0 (ATCC #CRL 8287). SP2/0 cells produce only immunoglobulin encoded by the transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid. Other suitable recipient cells include lymphoid cells such as B lymphocytes of human or non-human origin, hybridoma cells of human or non-human origin, or interspecies heterohybridoma cells. The expression vector carrying a chimeric antibody construct of the present invention may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium, phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment. The chimeric immunoglobulin genes of the present invention can also be expressed in nonlymphoid mammalian cells or in other eukaryotic cells, such as yeast, or in prokaryotic cells, in particular bacteria. Yeast provides substantial advantages over bacteria for the production of Ig H and L chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies now exist which utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre- peptides). Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and the stability of chimeric H and L chain proteins and assembled chimeric antibodies. A__αy of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. A number of approaches may be taken for evaluating optimal expression plasmids for the expression of cloned immunoglobulin cDNAs in yeast (see Glover, D.M., ed., DNA Cloning, IRL Press, 1985). Bacterial strains may also be utilized as hosts for the production of antibody molecules or antibody fragments described by this invention., E. coli K12 strains such as E. coli W3110 (ATCC 27325), and other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may be used. Plasmid vectors containing replicon and control sequences which are derived from, species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replica- tion site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches may be taken for evaluating the expression plasmids for the production of chimeric antibodies or antibody chains encoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, supra). Preferred hosts are mammalian cells, grown in vitro or in vivo. Mammalian cells provide post-translational modifications to im_munoglobulin protein molecules including leader peptide removal, folding and assembly of H and L chains, glycosylation of the antibody molecules, and secretion of functional antibody protein. Mammalian cells which may be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin, such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61). Many vector systems are available for the expression of cloned H and L chain genes in mammalian cells (see Glover, supra). Different approaches can be followed to obtain complete H₂L₂ antibodies. For in vivo use, particularly for injection into humans, it is desirable to decrease the immunogenicity of the mAb by making mouse-human (or rodent-human) chimeric antibodies as above, or by humanizing the antibodies using methods known in the art. The humanized antibody may be the product of an animal having transgenic human Ig Constant region genes (see for example WO 90/10077 and WO 90/04036). Alternatively, the antibody of interest may be genetically engineered to substitute the CH], CH₂, CH₃, hinge domains, and/or the framework domain with the corresponding human sequence (see WO 92/02190). Single Chain Antibodies The antibody of the present invention may be produced as a single chain antibody or scFv instead of the normal multimeric stmcture. Single chain antibodies include the hypervariable regions from an Ig of interest and recreate the antigen binding site of the native Ig while being a fraction of the size of the intact Ig (Skerra, A. et al. (1 88) Science, 240: 1038-1041; Pluckthun, A. et al. (1989) Methods Enzymol 178: 497-515; Winter, G. et al. (1991) Nature, 349: 293-299); Bird et al, (1988)

Science 242:423; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879; Jost CR et al,. JBiol Chem. 1994262:26267-26273; U.S. Patents NTo. 4,704,692, 4,853,871, 4,94,6778, 5,260,203, 5,455,030). DNA sequences encoding the V regions of the H chain and the L chain are ligated to a linker encoding at least about 4 amino acids (typically small neutral amino acids). The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody. One method of producing the antibodies of the present invention is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9- fluorenylmethyloxycarbonyl) or tBoc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to an antibody chain or antigen-binding fragment thereof can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized but not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their C- and N- termini, respectively, to> form an antibody, or a fragment thereof. (Grunt, GA, Synthetic Peptides: A User Guide, W.H. Freeman, and Co., N.Y. (1992); Bodansky, M et al, eds, Principles of Peptide Synthesis, Springer-Verlag Inc ., N.Y. (1993)) Antibodies can be selected for particular desired properties. In the case of an antibody to be used in vivo, antibody screening procedures can include any of the in vitro or in vivo bioassays that measure binding to parafibromin, e.g., to cells expressing this protein. Moreover, the antibodies may be screened in various of tumor models such as a xenogeneic mouse model in which a human parathyroid carcinoma line is grown in immunocoirrpromised, e.g., nude, mice. Diagnostically Labeled Antibody The term "diagnostically labeled" means that the present antibody has attached to it a detectable label that is diagnostically useful. There are many different labels and methods of labeling known to those of ordinary skill in the art, described below. General classes of labels which can be used in the present invention include radioactive isotopes, paramagnetic is topes, and compounds which can be imaged by positron emission tomography (PET), fluorescent o_τ colored compounds, etc. Suitable detectable labels include radioactive, fluorescent, fluorogenic, chromogenic, or other chemical labels. Useful radiolabels (radionuclides), which are detected simply h y gamma counter, scintillation counter or autoradiography include ³H, ¹²⁵1, ^13II, ³⁵S and ¹⁴C ¹³¹I is also a useful therapeutic isotope (see below). A number of U.S. patents, incoφorated by reference herein, disclose methods and compositions for complexing metals to larger molecules, including description of useful chelating agents. The metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules. These documents include: U.S. Patents 5,627,286; 5,618,513; 5,567,408; 5,443,816; and 5,561,220. Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The fluorophore, such as the dansyl group, must be excited by light of a particular wavelength to fluoresce. See, for example, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR., 1996). Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon Green™ and its derivatives, Rhodamine Green™ and Rhodol Green™, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups. Similarly, fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziricline-reactive groups. The long wavelength rhodamines, which are basically Rhodamine Green™ derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known. Their spectra are not affected by changes in pH between 4 and 10, an important advantage over the fluoresceins for many biological applications. This group includes the tetramethylrhocdamines, X-rhodamines and Texas Red™ derivatives. Other preferred fluorophores for derivatizing the eptide according to this invention are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives. Also included as labels are two related inorganic materials that have recently been described: semiconductor nanocrystals, comprising, for example, cadmium sulfate (Bruchez, M. et al, Science 281:2013-2016 (1998), and quantum dots, e.g., zinc-sulfide-capped Cd selenide (Chan, W.C.W. et al, Science 281 :2016-2018 (1998)). In yet another approach, the amino group of the antibody is allowed to react with reagents that yield fluorescent products, for example, fluorescamine_^ dialdehydes such as o-phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3-dicaxboxylate. 7-nitrobenz-2-oxa-l,3-diazole (NBD) derivatives, both chloride and fluoride, are useful to modify amines to yield fluorescent products. The antibody of the invention can also be labeled for detection using fluorescence-emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the peptide using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A, see Example X, infra) or ethylenediaminetetraacetic acid (EDTA). DTP A, fo_r example, is available as the anhydride, which can readily modify the NH₂-containing peptides of this invention. For in vivo diagnosis or therapy, radionuclides may be bound to the antibody either directly or indirectly using a chelating agent such as DTPA and EDTA. Examples of such radionuclides are ⁹⁹Tc, ¹²³1, ¹²⁵1, ^13II, ^{n ι}In, ⁹⁷Ru, ⁶⁷Cu, ⁵⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹V and ²⁰¹T1. Generally, the amount of labeled antibody needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.01 mg/kg to 100 mg/kg. The antibody can also be made detectable by coupling to a phosphorescent or a chemiluminescent compound. The presence of the chemiluminescent-tagged peptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescers are liαminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the peptides. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for puφoses of labeling are luciferin, luciferase and aequorin. In yet another embodiment, colorimetric detection is used, based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients. In situ detection of the labeled peptide may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label. Those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. For diagnostic in vivo radioimaging, the type of^" detection instrument available is a major factor in selecting a radionuclide. The radionuclide chosen πrust have a type of decay which is detectable by a particular instrument. In general, any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention. Another factor in selecting a radionuclide for in vivo diagnosis is that its half-life be long enough so that the label is still detectable at thre time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized. In one preferred embodiment, a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras. In vivo imaging may be used to detect occult metastases which are not observable by other methods. Imaging could be used, for example, to stage tumors non-invasively.

Use of Antibodies to Detect Parafibromin by Immunoassay Antibodies specific for an epitope of parafibromin are useful in immunoas-says to detect molecules containing these epitopes in tissue sample or a body fluid, such as serum or plasma. Such antibodies would detect parafibromin or a epitope-bearing fragment thereof. Thus, if proteolysis in the tumor milieu results in release of the fragments or in tissue. Any conventional immunoassay known in the art may be employed for this puφose, though Enzyme Immunoassays such as ELISA are preferred. Immunoassay methods are also described in references cited above.

Immunohistochemical Assays A preferred assay for detecting parafibromin in a tissue is by immunohisto chemistry, using any conventional assay methods, with which the art is replete. A preferred assay is the; one described in the Examples below. For a description of such methods, see, for example, Dabbs, DJ, Diagnostic

Immunohistochemistry, Churchill Livingstone, 2001, which is incoφorated by reference in its entirety.

Non-Histological Immunoassays Preferred immunoassays are enzyme immunoassays (EIA's) such as ELIS.A, which employ antigens or antibodies immobilized to solid supports. For the present compositions and methods, the solid support is preferably any one of polystyrene, polypropylene, polyethylene, dextran, nylon, polyacrylamide, polyvinylidene difluoride, natural cellulose, modified cellulose, nitrocellulose, agarose and magnetic beads. In a preferred embodiment, the surface of polystyrene or other plastic multiwell plates serves as the solid support. In another embodiment, a solid support to which the antibody or antigen is affixed to the bottom or placed loosely in the wells of multiwell plates. _Multiwell plates in which the bottoms of the wells comprise nitrocellulose or a similar membrane material and through which liquid can be moved under pressure or vacuum may also be used. Typical, and preferred, immunoassays include "forward" assays in which the antibody immobilized to a solid support is first contacted with the sample being tested to bind or "extract" the antigen from the sample by formation of a binary immobilized antibody-antigen complex. After suitable incubation, the solid support is washed to remove the residue of the fluid sample including unbound antigen, if any, and then contacted with the solution containing an unlcnown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation that permits the labeled antibody to complex with the immobilized antigen through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody and the immobilized label is measured. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the amount of immobilized labeled antibody with the amount immobilized when a standard sample containing a known quantity of antigen is used. So called "simultaneous" and "reverse" sandwich assays may also be used. A simultaneous assay involves a single incubation step as the immobilized antibody and labeled antibody are added simultaneously to the sample. After appropriate incubation, the solid support is washed to remove residue of the sample and uncomplexed labeled antibody. The presence or amount of labeled antibody associated with the solid support is then determined as in the above conventional "forward" sandwich assay. In a "reverse" assay, a solution of labeled antibody is added to the sample after a suitable incubation period followed by addition of immobilized unlabeled antibody. After a second incubation, the solid phase material is washed in conventional fashion to free it of the residue of the sample and unreacted labeled antibody. The determination of immobilized antibody associated with the solid support is then determined as in the "simultaneous" and "forward" assays. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 Materials and Methods

Source of Specimens A retrospective multi-center study was conducted involving anonymized formalin-fixed, paraffin-embedded parathyroid specimens from patients with primary hypeφarathyroidism from eight institutions worldwide - Ohio State University, Leiden University Medical Center, Northwestern University, Seinajoki Central Hospital, Singapore General Hospital, National University Hospital

(Singapore), Shared Pathology Informatics Network (Michigan, USA) and University of Tasmania. The study was approved by the Van Andel Institute Institutional Review Board. Patient Samples A total of 160 specimens were examined. 123 full sections were studied, including sporadic primary carcinomas (n=19), sporadic metastatic tissue (n=l), sporadic equivocal carcinomas (n=2), sporadic adenomas (n=50), sporadic primary hypeφlasia (n=25), MEN 1 -associated tumors (n=13), HPT- JT carcinoma (n=l), HPT-JT adenomas (n=7) and normal tissue (n=5). 2 sporadic adenomas were subsequently classified after immunostaining as equivocal carcinomas, and calculations were performed as such. A tissue array containing an additional 37 specimens was also studied: sporadic primary carcinomas (n=23), sporadic metastatic tissue (n=7), sporadic equivocal carcinomas (n=3), HPT-JT carcinoma (n=l), HPT-JT adenomas (n=2) and normal tissue (n=l). 28 arrayed definite carcinoma specimens had been previously characterized (Haven et al, supra): 10/28 were cystic; 4/28 trabecular; 19/28 had fibrotic bands; 8/28 had >1/10 mitoses/HPF; 14/28 showed positive cyclin Dl staining; 20/27 showed positive CASR staining; 14/22 had loss of heterozygosity (LOH) of chromosome lq (HRPT2 gene loci); 13/22 had LOH at chromosome 1 lq (MEN1 gene loci); Ki-67 index ranged from 0.1 to 27.5. 1 arrayed specimen of metastatic tissue was subsequently demonstrated to be of non-parathyroid origin, and was excluded from all calculations. Lesions were previously diagnosed as definite carcinomas only if vascular invasion, invasion of surrounding tissue or distant metastasis were evident. Equivocal carcinomas were defined as tumors exhibiting histopathologic features of carcinoma without the presence of vascular invasion, invasion of surrounding tissue or distant metastasis. All HPT-JT adenomas had been previously sequenced (Caφten et al, supra; Haven, CJ et al, J Clin Endocrinol Metab, 2000. 85:1449-54) and confirmed to have HRPT2 gene mutations. All MEN1 tumors had been previously confirmed to have MEN1 gene mutations. hnmunohistochemistrv Immunohistochemistry was performed using a standard procedure. Deparaffinized 5μm sections were steamed in citrate buffer for 30 minutes. Sections were incubated in 0.3% hydrogen peroxide and 5% goat serum; in primary antibody (50 μg/mL) overnight at 4°C; followed by biotinylated goat anti- murine Ig antibody (6 μg/mL) for 60 minutes. Sections were incubated with avidin-biotinylated enzyme complex (Vector Laboratories, PK-6100) and subsequently with diaminobenzene substrate (Vector Laboratories, SK-4100). Positive controls were transfected COS7 cells expressing parafibromin. Slide sections were examined independently by two blinded investigators (CM. and J.R.), and the arrays were examined by two unblinded investigators (CM. and H.M.). Staining patterns were classified as diffusely positive, focal loss or diffuse loss. The intensity of staining was also evaluated, on a semi-quantitative scale of 0 to 3. Inter-observer agreement for blinded assessments was calculated using kappa statistics. EXAMPLE 2 Generation and Validation of anti-HRPT2 (anti-parafibromin) MAb The peptide RRPDRKDLLGYLNC (SEQ ID NO:4) was designed as the immunogen based on the HRPT2 sequence (SEQ ID NO:2). This peptide is preferred for the following reasons. First, it has a high degree of homology to the homologues of HRPT2 in Drosophila and C. elegans. Second, the N- terminal 7 residues are hydrophilic and therefore more likely to be exposed on the surface of the folded native protein. The Cys residue was added at the C-terminus as a site for coupling of the peptide to a carrier protein, such as KLH. This peptide was synthesized by Genemed Synthesis Inc. Briefly, the peptide was conjugated to KLH, and injected into BALB/c mice intraperitoneally (i.p.) in complete Freund's adjuvant (50 μg conjugate), followed by two additional injections of the same dose in incomplete Freund's adjuvant at two week intervals. After one month, a final injection was given i.p (50 μg in 0.5 ml PBS) and intravenously (i.v.) (50 μg in 0.2ml PBS) without adjuvant. Spleen cells were harvested three days after the final injection and fused with P3X63AF8/653 myeloma cells using standard techniques. HEK293 cells co-transfected with a GFP-HRPT2 DNA construct were used to test hybridoma supernatants for anti-HRPT2 activity. In such cells, GFP is localized in the nucleus, therefore, if the Rhodamine Red that was conjugated to the secondary ant-mouse Ig antibody ) was found co-localized with the GFP green fluorescence, the supernatant would be scored positive for a mAb would be HRPT2- specific. These transfected cells were plated onto ten of 96-well plates. The cells were fixed with methanol/acetone (50/50). Hybridoma supernatants were screened for reactivity to HRPT2 by immunohistochemical staining. Hybridoma supernatant (50μ) was added to wells containing fixed 293 cells for 1.5 h at 37°C. Plates were washed twice in washing buffer (PBS/0.05% Tween-20), and Rhodamine Red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Lab) was added (30 μl/well) at 1:100 dilution for 1.5 h at 37°C. After washing twice in washing buffer, cells were examined under fluorescence microscope. If a supernatants that showed positively staining (Rhodamine Red co-localized with GFP in the cell nucleus), the hybridoma clone would be selected, expanded and tested for reactivity to the immunizing peptide by ELISA. IHC staining was used to test hybridoma supernatants for the presence of anti-HRPT2 antibody. In one approach, the original cells were tagged with a GFP by transduction as above. The green fluorescence was visualized by confocal fluorescence imaging. The hybridoma supernatants with GFP fusion protein was applied and then washed off the sections/cells. The antibody that remained would express green fluorescence when a blue light (Argon laser line 488) was shined on the preparation. The green staining clearly identified positive cells. In this ELISA, the peptide was first coupled to the carrier protein OVA (coupling kit from Pierce Chemical Co., Cat# 0077608). The peptide/OVA conjugate was coated onto wells of 96 well EIA plates (2μg/ml in 50μ per well) in coating buffer (0.2 M Na₂C0₃/NaHC0₃, pH9.6). Plates were incubated overnight at 4 °C and then blocked with a PBS solution containing 1% BSA (200 μl/well) overnight at 4°C 50μl of hybridoma supernatants were added to wells for 1.5 hours at RT. Plates were washed twice in washing buffer (PBS/0.05% Tween-20), and alkaline phosphatase coupled goat-anti-mouse IgG (Sigma, St. Louis, MO) was added (50 μl/well) at a 1:2000 dilution and allowed to incubate for 1.5 hours at RT. After washing 4X in washing buffer, the chromogenic substrate CP-nitrophenylphosphate (Kirkegaard & Perry Co., Gaithersburg, MD), was added for 30 min and absorbance measured at 405nm. Hybridomas with strong reactivity with HRPT2 peptide ( ^os >1.0; negative controls <0.02) were re- cloned twice, and reactivity was again confirmed by ELISA as above. A positive Clone 2H1 was recloned twice, and the mAb was produced and FPLC-purified using a protein-G affinity column. This 2HlmAb was characterized as a mouse IgGl/κ antibody. EXAMPLE 3 2H1 MAb Distinguishes Parathyroid Carcinoma Parathyroid carcinoma was distinguished from other benign pathologies by the loss of parafibromin nuclear immunoreactivity. Table 1 summarizes the descriptive evaluation of the results with the tie-breaking evaluation included. Eleven benign cases from the adenoma, hypeφlasia and MEN1 -tumor groups displayed heterogeneity of staining without absence of parafibromin immunoreactivity, and these were classified as diffusely positive. The assay had a calculated sensitivity of 96% (95% CI. 85% - 99%) and specificity of 99% (95% CI. 92% - 100%) for differentiating parathyroid carcinoma from sporadic benign proliferation (Table 2). Based on a common estimate of a prevalence of parathyroid carcinoma in primary hypeφarathyroidism of 1% (Grimelius et al, supra; Newcombe, RG, StatMed, 1998. 17: 857-72), the positive predictive value (PPV) would be an estimated 49% (95% CI. 10% - 100%), and the negative predictive value (NPV) would be almost 100% (95% CL 100% - 100%, with rounding). In countries such as Japan and Italy (Grimelius et al., supra; Obara, T et al., Endocrinol Jpn, 1989. 36: 135-45) with an estimated prevalence of 5%, the PPV and NPV would be 83% (95% CI. 36% - 100%) and 99% (95% CI. 99% - 100%o) respectively. The data showing inter-observer variation is presented in Table 3. There was exceptional inter- observer agreement with regards to the blinded assessment of any immunoreactivity loss, with an unweighted kappa statistic of 0.89 (95% CI. 0.79 -0.98). Agreement with regards to staining pattern was also excellent, with an unweighted kappa statistic of 0.77 (95% CL 0.65-0.89). The hematoxylin-eosin stained sections of benign specimens which had at least one investigator assess as having loss of parafibromin immunoreactivity (n=6) were re-evaluated by two pathologists. Two adenomas from separate institutions received revised diagnosis of equivocal, but highly probable, carcinoma based on severe architectural atypia, nuclear atypia and abundant mitotic figures, but lacking the criteria of invasion or metastasis. In addition, one specimen, diagnosed previously as metastatic carcinoma, demonstrated diffuse nuclear immunoreactivity and was demonstrated by parathyroid hormone immunostaining (Sawady, J et al., Mod Pathol, 1989. 2: 652-657) to be of non-parathyroid origin. All but one HPT-JT adenomas exhibited loss of parafibromin immunoreactivity, the remaining one evaluated as diffuse weak staining by one investigator, and by the other as diffuse loss.

Ranges reflect discordance in number of diagnoses by the independent blinded investigators for the tissue sections TABLE 2: Calculated Diagnostic Value Indices

TABLE 3: Results of blinded individual observer evaluation for all sections (n=123)

The loss of parafibromin occurred regardless of tissue architecture, histopathologic features and LOH at the HRPT2 and MEN1 loci. HPT-JT adenomas were been excluded from the analysis on basis of their malignant potential. The presence of strong parafibromin immunoreactivity highlighted one lymph node specimen in the anay, diagnosed previously as metastatic carcinoma. Parathyroid hormone immunostaining confirmed that this specimen was probably not parathyroid tissue. The results also localize human parafibromin to the nucleus. This nuclear localization is consistent with results of cellular fractionation studies and immunohistochemical analysis of human and murine tissue. Parafibromin shares 32% amino acid sequence identity with a yeast protein cdc73p, which is also a nuclear protein and part of the Pafl complex involved in cell cycle regulation and transcription. Patients with HPT-JT syndrome have a high risk of parathyroid carcinoma (Haven CJ et al, (in press) supra. In conjunction with microarray studies showing clustering of parathyroid carcinomas and HPT-JT-related adenomas (Haven et al. (in press) supra, the shared loss of parafibromin immunoreactivity in both pathologies suggests that this is an early and pivotal step in parathyroid tumorigenesis. In summary, the discovery that a loss of parafibromin in parathyroid carcinoma but not in benign lesions is the basis for a method based on parafibromin immunostaining that permits the distinguishing of parathyroid carcinoma. DISCUSSION OF EXAMPLES 1-3 This is the first study on parafibromin, the protein product of the HRPT2 tumor suppressor gene. The results localize human parafibromin to the nucleus, which is consistent with cellular fractionation studies, and immunohistochemistry in a variety of tissues. Parafibromin shares 32% identity with yeast protein cdc73p (Carpten et al., supra), which is also a nuclear protein and part of the Pafl complex mediating cell cycle regulation and transcription (Shi, X et al., Mol Cell Biol, 1997. 17: 1160-69). Its function in humans is currently unknown. Pathology Assessment This study demonstrates that parafibromin immunoreactivity is a useful adjunct for differentiating carcinoma from benign tissue. Parathyroid carcinoma is often not recognized, even after histological examination. With the approximately 20% (3/15) possibility that apparently sporadic parathyroid carcinoma may be a manifestation of hereditary HPT-JT syndrome, making an accurate diagnosis is now paramount. To the present inventors' knowledge, no other immunohistochemical marker for parathyroid carcinoma has been previously disclosed in a study of similar size or geographical diversity. The loss of parafibromin immunoreactivity was true regardless of architecture, presence of mitotic figures, loss of heterozygosity and immunostaining for CASR and cyclin Dl. In addition, this study successfully detected the misclassification of two adenomas among fifty cases, demonstrating that parathyroid malignancy is often under-recognized. Only one other study reported a re-classification of parathyroid tumors (Levin, KE et al, J Clin Endocrinol Metab, 1988. 67:779-84), based on using DIMA cytometry, two 'atypical adenomas' (or equivocal carcinomas) that subsequently relapsed were classified as 'carcinomas.' Aneuploidy as demonstrated on DNA cytometry is, however, of prognostic rather than diagnostic significance (Shattuck et al, supra; Levin et al, supra; Bondeson et al. , supra; August et al. , supra; Haven et al. , 2000, supra). In assessing the value of a diagnostic assay, the PPV and NPV are the most helpful parameters (Jaeschke, R et al., JAMA, 1994. 271:703-7); these depend on the prevalence of a disease within a certain population, as well as the sensitivity and specificity. With an estimated prevalence of 1% of cases, this assay yields a PPV of 49% (95% CL 10% -100%) and an NPV of 100% (95% CI. 100%, - 100%, with rounding) In countries with a prevalence of 5%, such as Japan and Italy, the PPV and NPV would be 83% (95% CL 36% - 100%) and 99% (95% CL 99% - 100%) respectively. Based on the foregoing, parafibromin immunostaining will be a helpful diagnostic adjunct for the pathologist. Adenomas with HRPT2 mutations also demonstrate diffuse loss of parafibromin immunoreactivity in our study, these are of very low frequency in sporadic parathyroid adenomas.

However, the loss of parafibromin immunoreactivity is certainly useful in detecting these tumors. For patients with HPT-JT syndrome who are at high risk of carcinoma, deliberate total prophylactic parathyroidectomy has been considered (Jaeschke, R et al, JAMA, 1994. 271 : 703-7). Whether radical surgery will benefit the rare patient who has had a somatic HRPT2 mutation detected in a parathyroid adenoma remains uncertain. While definite carcinomas may be recognized on the definitive criteria of invasion or metastasis, tumors that have histopathologic features of malignancy but lack tumor extension represent challenging clinical and pathological problems. Levin (supra) distinguished between "typical" and "atypical adenomas." Others have chosen to label this group as "equivocal carcinomas" (Erickson, LA et al, 1999, supra Bondeson et al, supra). As the histopathological difference between an "atypical adenoma", an "equivocal carcinoma" and a localized parathyroid carcinoma remains unclear, it may be helpful to categorize this group as "equivocal carcinomas" for the purposes of research and follow-up. Considering these group of tumors as "equivocal carcinomas" is also clinically logical, since patients with these tumors are followed up carefully with serum calcium and PTH testing for a possible relapse, in a similar fashion to patients with parathyroid carcinoma (Levin et al, supra; Goshen, O et al, J Laryngol Otol, 2000. 114:302-4. Finally, the foregoing may be more appropriate in view of the potential malignant behavior of these group of tumors, the under-recognition of parathyroid carcinoma and the fact that current gold- standard diagnostic criteria of malignancy of invasion or metastasis is limited to advanced disease. These results support the view that the entity termed "equivocal carcinoma" is fundamentally heterogeneous and intermediate between the carcinoma and the benign groups (Levin, KE et al, Surgery, 1987 101:649-60. Of the five equivocal cases examined, three displayed loss of parafibromin immunoreactivity. In one of these three cases, no relapse of carcinoma was detected after five years of follow-up. In the other two cases showing loss of immunoreactivity, no follow-up was available, but the diagnosis of carcinoma in these two cases was strongly supported on clinical grounds. In the 2 equivocal cases that stained positive, no relapse was detected during follow-up of 3 and 7 years respectively. The evaluation of immunohistochemically stained slides has an is inherently subjective component. Although the diagnoses described herein were blinded, vascular or local tissue invasion may have been visible on examination. However, any biases are unlikely to be significant because the vast majority of benign cases stained with either moderate or strong intensity, rather than weak staining (Table 1). Adenomas with HPT-JT mutations were uniformly diagnosed as loss of parafibromin immunoreactivity, whereas adenomas without these mutations were not.

Parafibromin and HRPT2 Gene Mutation 49 of 51 definite parathyroid carcinomas displayed loss of parafibromin immunoreactivity. This is consistent with the high rate of somatic HRPT2 gene mutations with biallelic inactivation reported in sporadic parathyroid carcinoma. Gene regulatory mechanisms may also account for part of this phenomenon. It is interesting that a small subset should display normal parafibromin expression. This may be due to alternative tumorigenic mechanisms; there is evidence that at least one additional tumor suppressor gene may exist on chromosome 13q. A recent study of two such candidate genes KB and BRCA2 did not identify any mutations in 7 specimens of parathyroid carcinoma (Shattuck, supra). Additionally, two previous case reports have documented that parathyroid carcinoma is a rare manifestation in MEN1 (Dionisi, S et al,., Mayo Clin Proc, 2002. 77:866-9; Sato, M et al, Endocrine, 2000. 12:223-6). The present results show that parafibromin expression is unimpaired in MEN1 -related benign tumors. HPT-JT syndrome is characterized by HKPT2 gene mutations, and patients with HPT-JT syndrome have a high risk of parathyroid carcinoma. Approximately 15% of parathyroid tumors are malignant (Marx et al, supra). In conjunction with microarray studies that showed a common gene signature for parathyroid carcinomas and HPT-JT-related adenomas, the loss of parafibromin immunoreactivity in both groups suggests that parafibromin downregulation is an early and pivotal step in parathyroid tumorigenesis. The present results link the loss of parafibromin immunoreactivity to the HRPT2 gene mutation in adenomas, and to the microarray gene signature. Given the high prevalence of HRPT2 gene mutations in parathyroid carcinoma, the same loss of parafibromin immunoreactivity is likely to be related to HRPT2 gene mutations. It is interesting to note that focal expression was not lost in a small subset of parathyroid adenomas with documented HRPT2 gene mutations. In tumors with focal loss of parafibromin immunoreactivity, parafibromin expression was markedly higher near blood vessels and margins (both internal and external) such as fibrous septa and capsular tissue. The antibody may have bound to wild- type or mutant parafibromin. Since en-bloc resection is the definitive therapy for parathyroid carcinoma, the present invention may be extended to intra-operative assessment of parafibromin immunoreactivity through the use of ultrarapid immunostaining (Richter, T et al, J Clin Pathol, 1999. 52:461-3). Given that up to 86% of carcinomas are not detected on intraoperative diagnosis (Hundahl, SA et al, 1999. 86:538-44), this would be of significant benefit. EXAMPLE 4 Assessment of HRPT2 in Clear Cell-Renal Cell Carcinoma and Bladder Cancer Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, representing 2% of all malignancies and 2% of cancer-related deaths (Chow et al., JAMA 1999; 281:1628-31). Classification and histopathology is described by Mostfi, FK et. al, 1998, WHO International Histological Classification of Kidney Tumors and in Sobin, LH et al, eds., TNM classification of malignant tumors. 5th ed. (John Wiley & Sons, New York 1997). Renal cell carcinomas include several distinct entities with a range of biologic and clinical behavior from relatively indolent to extremely aggressive. The most widely used tumor-related prognostic factors include stage, grade, and histologic type. Traditional prognostic factors remain the most valuable (Gelb AB, Cancer, 1997, 80:981-986) Two discrete entities of superficial bladder cancer exist, one a low-grade innocuous tumor and the other a high-grade potentially lethal tumor, and these vary in their histologic appearance, risk of tumor recurrence, pattem of recurrence, and risk of tumor progression. Currently there are no sufficiently reliable prognostic markers so patients with a higher risk of tumor recurrence or progression are identified only by clinical factors (tumor stage, tumor grade, number of tumors (multifocality), etc. (Donat SM, Urol Clin North Am. 2003, 30:765-76). The present inventors show that parafibromin expression may be a useful new prognostic marker. Samples of tissue from CC-RCC and bladder carcinoma were tested by IHC for expression of parafibromin antigen as described above. The results are shown in Table 4 along with the a pathology score at the time of surgery (Tumor grade) which is correlated with severity and prognosis (see references cited above). The results show that the vast majority of these tyhpes of cancers show reduced expression of the HRPT2 gene product, parafibormin. The lower the expression, the higher is the grade of the tumor, indicating a poorer patient prognosis. Thus these findings are essentially the same as those for parathyroid tumors presente in Examples 1-3. Similar results are being obtained with lung, breast prostate and colorectal carcinomas, wherein parafibromin expression levels are decreased in the tumors compared to nonnal tissue, and the degree of lowered expression correlates with a poorer prognosis. Table 4: Assessment of HRPT2 in clear cell renal cell carcinoma and Bladder Cancer

All the references cited above are incoφorated herein by reference in their entirety, whether specifically incorporated or not. Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

Claims

1. An antibody specific for an epitope of human parafibromin.

2. The antibody of claim 1 which is specific for an epitope the expression, or detectable level of which, is reduced in or on a carcinoma cell or tissue compared to a normal cell or tissue or other non-carcinoma human cell or tissue.

3. The antibody of claim 2 wherein said expression or detectable level is reduced in or on parathyroid carcinoma, bladder carcinoma or renal carcinoma cells or tissue compared to normal human parathyroid, bladder or renal cells or tissue, respectively.

4. The antibody of claim 3 wherein said expression or detectable level is reduced in or on parathyroid carcinoma cells or tissue compared to normal human parathyroid cells or tissue.

5 The antibody of any of claims 1-4 wherein said epitope is formed by, or comprises, a peptide having the sequence RRPDRKDLLGYLN (SEQ ID NO:3).

6. The antibody claim 5 wherein said epitope is formed by a peptide having the sequence

RRPDRKDLLGYLN (SEQ ID NO:3).

7. The antibody of any of claims 1 -6 which is a monoclonal antibody (mAb).

8. The antibody of claim 7 which is murine mAb 2H1 deposited in the ATCC under accession number or a mAb antibody that binds specifically to the same parafibromin epitope as that bound by mAb 2H1.

9. The antibody of claim 8 which is murine mAb 2H1 deposited in the ATCC under accession number .

10. The antibody of claim 7 which is a chimeric mAb.

11. The antibody of claim 7 which is a human or humanized mAb.

12. The antibody of any of claims 1-11 immobilized to a solid support.

13. A diagnostically useful antibody according to any of claim 1-12 which is detectably labeled with a diagnostically useful label.

14. A diagnostically useful antibody composition comprising: (a) the labeled antibody of claim 13; and (b) a diagnostically acceptable carrier or diluent.

15. The composition of claims 8 wherein the label is a radionuclide, a PET-imageable agent, an MRI-imageable agent, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer.

16. The composition of claim 15, wherein the label is a radionuclide selected from the group consisting of ³H, ¹⁴C, ³⁵S, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁷Ru, ⁹⁹Tc, ^mIn, ^,231, ¹²⁵I, ^,311, ¹⁶⁹Yb and ²⁰¹T1.

17. The composition of claims 15 wherein the label is a fluorescer or fluorogen selected from the group consisting of fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green and Texas Red.

18. A method for producing a hybridoma cell line that produces a mAb specific for an epitope of human parafibromin, said method comprising: (a) immunizing a donor animal with a parafibromin peptide or polypeptide conjugated to a earner; (b) obtaining B lymphocytes from said immunized donor animal; (c) fusing said B lymphocytes with cells of a fusion partner cell line to obtain hybrid cells; (d) selecting hybrid cells which produce the parafibromin-specific antibody by: (i) culturing said hybrid cells; (ii) screening the medium of said cultures for the presence of an antibody which binds to parafibromin in an immunoassay or to cells expressing parafibromin in an immunohistochemical assay, thereby detecting antibody producing hybrid cells, and (e) growing said selected hybrid cells, thereby producing said hybridoma cell line.

19. A method for producing a mAb specific for an epitope of human parafibromin, comprising (a) culturing a hybridoma cell line produced in accordance with claim 18 under conditions which permit antibody production and secretion by said cell line; (b) obtaining the culture medium containing said antibody; and (c) optionally, purifying said antibody.

20. The method of claim 18 or 19 wherein said epitope is one formed by, or which comprises, the amino acid sequence RRPDRKDLLGYLN (SEQ ID NO:3).

21. The method of claim 20 wherein said epitope is formed by amino acid sequence RRPDRKDLLGYLN (SEQ ID NO:3).

22. An immunoassay method for detecting carcinoma cells or tissue in a test sample suspected of containing carcinoma cells or tissue, comprising: (a) contacting said test sample with the antibody of any of claims 1-13 and (b) measuring the amount of antibody bound to the test sample compared to a control cell or tissue sample, wherein a decline or loss of parafibromin immunoreactivity in the test sample compared to the control is indicative of carcinoma.

23. The immunoassay method of claim 22 for detecting a parathyroid, bladder or kidney carcinoma in the test sample suspected of containing parathyroid, bladder or kidney carcinoma cells, wherein detection of a decline or loss of parafibromin immunoreactivity in the test sample compared to a control normal parathyroid, bladder or kidney sample, respectively, is indicative of said parathyroid, bladder or kidney carcinoma.

24. The method of claim 23 for detecting a parathyroid carcinoma in the test sample suspected of containing parathyroid carcinoma cells or tissue, wherein detection of a decline or loss of parafibromin immunoreactivity in the test sample compared to a control normal parathyroid sample is indicative of said parathyiOid carcinoma.

25. An immunoassay method for detecting a carcinoma in a test sample suspected of containing carcinoma cells or tissue, comprising: (a) contacting said sample with the diagnostic composition of any of claims 14-17; and (b) measuring the amount of antibody in said composition bound to the test sample compared to a control cell or tissue sample, wherein a decline or loss of parafibromin immunoreactivity in the test sample compared to the control is indicative of carcinoma.

26 The immunoassay method of claim 25 for detecting a parathyroid, bladder or kidney carcinoma in a test sample suspected of containing said parathyroid, bladder or kidney carcinoma, wherein detection of a decline or loss of parafibromin immunoreactivity in the test sample compared to a control normal parathyroid, bladder or kidney sample, respectively, is indicative of said parathyroid, bladder or kidney carcinoma.

27. The method of claim 19 wherein the immunoassay detects said parathyroid carcinoma in the test sample suspected of containing said parathyroid carcinoma, wherein detection of a decline or loss of parafibromin immunoreactivity in the test sample compared to a control normal parathyiOid sample is indicative of said parathyroid carcinoma .

28. The immunoassay method of any of claims 22-27 which is an immunohistochemical assay wherein the test and control samples are tissues and the immunoreactivity is detected as staining of the tissue.

29. The method of claim 28 wherein said staining is produced by a detectably labeled ligand for said antibody.

30. The method of claim 29 wherein the ligand is a secondary anti-immunoglobulin antibody.

31. An imaging method for detecting carcinoma in a subject, comprising: (a) administering to the subject the antibody of claim 13 or the composition of any of claims 14-17, wherein the antibody is labeled with an imageable label; and (b) measuring by an imaging technique the accumulation of the antibody in a tissue or region in which carcinoma is suspected, and comparing said accumulation to the antibody accumulation in unaffected tissue, wherein a decrease in antibody accumulation the suspected tissue or region compared to the unaffected tissue is indicative of the carcinoma.

32. The imaging method of claim 31 for detecting parathyiOid, bladder or kidney carcinoma in a subject, wherein the accumulation of the antibody is measured in a tissue or region in which parathyiOid, bladder or kidney carcinoma is suspected, and said accumulation is compared to the accumulation of antibody in unaffected parathyroid, bladder or kidney tissue, respectively, wherein a decrease in antibody accumulation the suspected tissue or region compared to the unaffected tissue is indicative of parathyroid, bladder or kidney carcinoma.

33. The imaging method of claim 32 wherein the carcinoma is parathyroid carcinoma and the unaffected tissue is parathyroid tissue.

34. A kit useful in a method of measuring or detecting parafibromin immunoreactivity in a sample, the kit being adapted to receive therein one or more containers, the kit comprising: (a) the antibody of any of claims 1-13, in detectably labeled form (b) optionally, one or more additional reagents useful for carrying out an immunoassay.

35. A kit useful in a method of measuring or detecting parafibromin immunoreactivity in a sample, the kit being adapted to receive therein one or more containers, the kit comprising: (a) the composition of any of claims 14-17, and (b) optionally, one or more additional reagents useful for carrying out an immunoassay.

36. The kit of claim 34 or 35 wherein the antibody is directly labeled with a radionuclide, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer.

37. A kit useful in a method of measuring or detecting parafibromin immunoreactivity in a sample, the kit being adapted to receive therein one or more containers, the kit comprising: (a) as a first antibody, the antibody of any of claims 1-11, and (b) one or more additional reagents useful for carrying out an immunoassay, which include a detectably labeled ligand for the first antibody.

38. The kit of claim 37 wherein the ligand is a labeled second antibody specific for the first antibody.