Pro115 Antibody Compositions And Methods Of Use Patent Application (2025)

U.S. patent application number 12/374292 was filed with the patent office on 2010-06-17 for pro115 antibody compositions and methods of use. This patent application is currently assigned to DIADEXUS, INC.. Invention is credited to Gundo Diedrich, Paul Levi Miller, Jackie Papkoff, Astrid Strelow.

PRO115 ANTIBODY COMPOSITIONS AND METHODS OF USE

Abstract

The invention provides isolated anti-Pro115 antibodies that bindto Pro115. The invention also encompasses compositions comprisingan anti-Pro115 antibody and a carrier. These compositions can beprovided in an article of manufacture or a kit. Another aspect ofthe invention is an isolated nucleic acid encoding an anti-Pro115antibody, as well as an expression vector comprising the isolatednucleic acid. Also provided are cells that produce the anti-Pro115antibodies. The invention encompasses a method of producing theanti-Pro115 antibodies. Other aspects of the invention are a methodof killing an Pro115-expressing cancer cell, comprising contactingthe cancer cell with an anti-Pro115 antibody and a method ofalleviating or treating an Pro115-expressing cancer in a mammal,comprising administering a therapeutically effective amount of theanti-Pro115 antibody to the mammal.

 LICATA & TYRRELL P.C. 66 E. MAIN STREET MARLTON NJ 08053 US

Related U.S. Patent Documents

Claims

1. An isolated antibody which competes for binding to the sameepitope as the epitope bound by the monoclonal antibody produced bya hybridoma, or subclone thereof, selected from the group ofPro115.A1, Pro115.A2, Pro115.A3, Pro115.A4, Pro115.A5, Pro115.A6,Pro115.A7, Pro115.A8, Pro115.A9, Pro115.A10, Pro115.A11,Pro115.A12, Pro115.A13, Pro115.A14, Pro115.A15, Pro115.A16,Pro115.A17, Pro115.A18, Pro115.A19, Pro115.A20, Pro115.A21,Pro115.A22, Pro115.A23, Pro115.A24, Pro115.A25, Pro115.A101.1,Pro115.A102.1, Pro115.A103.1, Pro115.A104.1, Pro115.A106.1,Pro115.A107.1, Pro115.A108.1, Pro115.B1, Pro115.B2, Pro115.B3,Pro115.B4, Pro115.B5, Pro115.B6, Pro115.B7, Pro115.B8, Pro115.B9,Pro115.B10, Pro115.B11, Pro115.B12, Pro115.B13, Pro115.B14,Pro115.B15, Pro115.B16, Pro115.B17, Pro115.B18, Pro115.B19,Pro115.B20, Pro115.B21, Pro115.B22, Pro115.B23, Pro115.B24,Pro115.B25, Pro115.B26, Pro115.B27, Pro115.B28, Pro115.B29,Pro115.B30, Pro115.B31, Pro115.B32, Pro115.B33, Pro115.B34,Pro115.B35, Pro115.B36, Pro115.B37, Pro115.B38, Pro115.B39,Pro115.B40, Pro115.B41, Pro115.B42, Pro115.B43, Pro115.B44,Pro115.B45, Pro115.B46, Pro115.B47, Pro115.B48, Pro115.B49,Pro115.B50, Pro115.B51, Pro115.B52, Pro115.B53, Pro115.B54,Pro115.B55, Pro115.B56, Pro115.B57, Pro115.B58, Pro115.B59,Pro115.B60, Pro115.B61, Pro115.B62, Pro115.B63, Pro115.B64,Pro115.B65, Pro115.B66, Pro115.B67, Pro115.B68, Pro115.B69,Pro115.D1, Pro115.D2, Pro115.D3, Pro115.D4, Pro115.D5, Pro115.D6,Pro115.D7, Pro115.D8, Pro115.D9, Pro115.D10, Pro115.D11,Pro115.D12, Pro115.D13, Pro115.F2 and Pro115.F3.

2. The antibody of claim 1 which competes for binding to the sameepitope as the epitope bound by the monoclonal antibody produced bya hybridomas Pro115.B7.1 or Pro115.B34.1.

3. The antibody of claim 1 which is a monoclonal antibody, anantibody fragment, a chimeric antibody, a human antibody orhumanized antibody.

4-5. (canceled)

6. The antibody of claim 1 which binds a Pro115 peptide consistingof Trp106 to Gly492 of Pro115.

7. The antibody of claim 6 wherein the Pro115 peptide contains apost translational modification, motif, or domain.

8. The antibody of claim 7 wherein the post translationalmodification, motif, or domain is a LDL receptor region, scavengerreceptor cys-region or a serine protease domain.

9. The antibody of claim 1 where the antibody competes for bindingwith a molecule which binds the LDL receptor region, scavengerreceptor cys-region or serine protease domain.

10. The antibody of claim 3 which competes for binding to the sameepitope as the epitope bound by the monoclonal antibody produced bya hybridoma selected from the group consisting of American TypeCulture Collection accession number PTA-7604 and PTA-7605 or whichis produced by a hybridoma selected from the group consisting ofAmerican Type Culture Collection accession number PTA-7604 andPTA-7605.

11. (canceled)

12. The antibody of claim 3 which is conjugated to a growthinhibitory agent or a cytotoxic agent.

13. (canceled)

14. The antibody of claim 13 claim 12 wherein the cytotoxic agentis selected from the group consisting of toxins, antibiotics,radioactive isotopes and nucleolytic enzymes.

15. (canceled)

16. The antibody of claim 14, wherein the toxin is selected fromthe group consisting of ricin, saponin, maytansinoid andcalicheamicin.

17. (canceled)

18. The antibody of claim 1 where the antibody inhibits the growthof Pro115-expressing cancer cells.

19-21. (canceled)

22. The antibody of claim 18, wherein the cancer cells are from acancer selected from the group consisting of prostate, colon, lungand pancreas cancer.

23. A cell that produces the antibody of claim 1.

24. The cell of claim 23, wherein the cell is selected from thegroup consisting of a hybridoma selected from the group consistingof American Type Culture Collection accession number PTA-7604 andPTA-7605.

25. (canceled)

26. A composition comprising the antibody of claim 1 and acarrier.

27-28. (canceled)

29. The composition of claim 26, wherein the antibody is anantibody fragment, a monoclonal antibody, a chimeric antibody, ahuman antibody or humanized antibody and the carrier is apharmaceutical carrier.

30. The composition of claim 29, wherein the humanized antibody isa humanized form of an anti-Pro115 antibody produced by hybridomaselected from the group consisting of American Type CultureCollection accession number PTA-7604 and PTA-7605.

31-47. (canceled)

48. An article of manufacture comprising a container and acomposition contained therein, wherein the composition comprises anantibody of claim 1.

49-72. (canceled)

Description

[0001] This patent application claims the benefit of priority fromU.S. Provisional Patent Application Ser. No. 60/832,551, filed Jul.21, 2006, teachings of which are herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to anti-Pro115 antibodycompositions and methods of killing Pro115-expressing prostate,colon, lung or pancreas cancer cells.

BACKGROUND OF THE INVENTION

Prostate Cancer

[0003] Prostate cancer is the most prevalent cancer in men and isthe second leading cause of death from cancer among males in theUnited States. AJCC Cancer Staging Handbook 203 (Irvin D. Fleminget al. eds., 5.sup.th ed. 1998); Walter J. Burdette, Cancer:Etiology, Diagnosis, and Treatment 147 (1998). The American CancerSociety estimated there will be 234,460 new cases of prostatecancer and 27,350 deaths in 2005. Additionally, the rate ofprostate cancer deaths in the United States for 1997-2001 was 31.5per 100,000 men, second only to lung and bronchus cancer. AmericanCancer Society website: cancer with the extension .org of the worldwide web. Cancer of the prostate typically occurs in older males,with a median age of 74 years for clinical diagnosis. Burdette,supra at 147. A man's risk of being diagnosed with invasiveprostate cancer in his lifetime is one in six. Platz et al., supraat 21.

[0004] Although our understanding of the etiology of prostatecancer is incomplete, the results of extensive research in thisarea point to a combination of age, genetic andenvironmental/dietary factors. Platz et al., supra at 19; Burdette,supra at 147; Steven K. Clinton, Diet and Nutrition in ProstateCancer Prevention and Therapy, in Prostate Cancer: aMultidisciplinary Guide 246-269 (Philip W. Kantoff et al. eds.1997). Broadly speaking, genetic risk factors predisposing one toprostate cancer include race and a family history of the disease.Platz et al., supra at 19, 28-29, 32-34. Aside from thesegeneralities, a deeper understanding of the genetic basis ofprostate cancer has remained elusive. Considerable research hasbeen directed to studying the link between prostate cancer,androgens, and androgen regulation, as androgens play a crucialrole in prostate growth and differentiation. Meena Augustus et al.,Molecular Genetics and Markers of Progression, in Management ofProstate Cancer 59 (Eric A Klein ed. 2000). While a number ofstudies have concluded that prostate tumor development is linked toelevated levels of circulating androgen (e.g., testosterone anddihydrotestosterone), the genetic determinants of these levelsremain unknown. Platz et al., supra at 29-30.

[0005] Several studies have explored a possible link betweenprostate cancer and the androgen receptor (AR) gene, the geneproduct of which mediates the molecular and cellular effects oftestosterone and dihydrotestosterone in tissues responsive toandrogens. Id. at 30. Differences in the number of certaintrinucleotide repeats in exon 1, the region involved intransactivational control, have been of particular interest.Augustus et al., supra at 60. For example, these studies haverevealed that as the number of CAG repeats decreases thetransactivation ability of the gene product increases, as does therisk of prostate cancer. Platz et al., supra at 30-31. Otherresearch has focused on the .alpha.-reductase Type 2 gene, the genewhich codes for the enzyme that converts testosterone intodihydrotestosterone. Id. at 30. Dihydrotestosterone has greateraffinity for the AR than testosterone, resulting in increasedtransactivation of genes responsive to androgens. Id. While studieshave reported differences among the races in the length of a TAdinucleotide repeat in the 3' untranslated region, no link has beenestablished between the length of that repeat and prostate cancer.Id. Interestingly, while ras gene mutations are implicated innumerous other cancers, such mutations appear not to play asignificant role in prostate cancer, at least among Caucasianmales. Augustus, supra at 52.

[0006] Environmental/dietary risk factors which may increase therisk of prostate cancer include intake of saturated fat andcalcium. Platz et al., supra at 19, 25-26. Conversely, intake ofselenium, vitamin E and tomato products (which contain thecarotenoid lycopene) apparently decrease that risk. Id. at 19,26-28 The impact of physical activity, cigarette smoking, andalcohol consumption on prostate cancer is unclear. Platz et al.,supra at 23-25.

[0007] Periodic screening for prostate cancer is most effectivelyperformed by digital rectal examination (DRE) of the prostate, inconjunction with determination of the serum level ofprostate-specific antigen (PSA). Burdette, supra at 148. While themerits of such screening are the subject of considerable debate,Jerome P. Richie & Irving D. Kaplan, Screening for ProstateCancer: The Horns of a Dilemma, in Prostate Cancer: AMultidisciplinary Guide 1-10 (Philip W. Kantoff et al. eds. 1997),the American Cancer Society and American Urological Associationrecommend that both of these tests be performed annually on men 50years or older with a life expectancy of at least 10 years, andyounger men at high risk for prostate cancer. Ian M. Thompson &John Foley, Screening for Prostate Cancer, in Management ofProstate Cancer 71 (Eric A Klein ed. 2000). If necessary, thesescreening methods may be followed by additional tests, includingbiopsy, ultrasonic imaging, computerized tomography, and magneticresonance imaging. Christopher A. Haas & Martin I. Resnick,Trends in Diagnosis, Biopsy, and Imaging, in Management of ProstateCancer 89-98 (Eric A Klein ed. 2000); Burdette, supra at 148.

[0008] Once the diagnosis of prostate cancer has been made,treatment decisions for the individual are typically linked to thestage of prostate cancer present in that individual, as well as hisage and overall health. Burdette, supra at 151. One preferredclassification system for staging prostate cancer was developed bythe American Urological Association (AUA). Id. at 148. The AUAclassification system divides prostate tumors into four broadstages, A to D, which are in turn accompanied by a number ofsmaller substages. Burdette, supra at 152-153; Anthony V. D'Amicoet al., The Staging of Prostate Cancer, in Prostate Cancer: AMultidisciplinary Guide 41 (Philip W. Kantoff et al. eds.1997).

[0009] Stage A prostate cancer refers to the presence ofmicroscopic cancer within the prostate gland. D'Amico, supra at 41.This stage is comprised of two substages: A1, which involves lessthan four well-differentiated cancer foci within the prostate, andA2, which involves greater than three well-differentiated cancerfoci or alternatively, moderately to poorly differentiated fociwithin the prostate. Burdette, supra at 152; D'Amico, supra at 41.Treatment for stage A1 preferentially involves following PSA levelsand periodic DRE. Burdette, supra at 151. Should PSA levels rise,preferred treatments include radical prostatectomy in patients 70years of age and younger, external beam radiotherapy for patientsbetween 70 and 80 years of age, and hormone therapy for those over80 years of age. Id.

[0010] Stage B prostate cancer is characterized by the presence ofa palpable lump within the prostate. Burdette, supra at 152-53;D'Amico, supra at 41. This stage is comprised of three substages:B1, in which the lump is less than 2 cm and is contained in onelobe of the prostate; B2, in which the lump is greater than 2 cmyet is still contained within one lobe; and B3, in which the lumphas spread to both lobes. Burdette, supra, at 152-53. For stages B1and B2, the treatment again involves radical prostatectomy inpatients 70 years of age and younger, external beam radiotherapyfor patients between 70 and 80 years of age, and hormone therapyfor those over 80 years of age. Id. at 151. In stage B3, radicalprostatectomy is employed if the cancer is well-differentiated andPSA levels are below 15 ng/mL; otherwise, external beam radiationis the chosen treatment option. Id.

[0011] Stage C prostate cancer involves a substantial cancer massaccompanied by extraprostatic extension. Burdette, supra at 153;D'Amico, supra at 41. Like stage A prostate cancer, Stage C iscomprised of two substages: substage C1, in which the tumor isrelatively minimal, with minor prostatic extension, and substageC2, in which the tumor is large and bulky, with major prostaticextension. Id. The treatment of choice for both substages isexternal beam radiation. Burdette, supra at 151.

[0012] The fourth and final stage of prostate cancer, Stage D,describes the extent to which the cancer has metastasized.Burdette, supra at 153; D'Amico, supra at 41. This stage iscomprised of four substages: (1) D0, in which acid phosphataselevels are persistently high, (2) D1, in which only the pelviclymph nodes have been invaded, (3) D2, in which the lymph nodesabove the aortic bifurcation have been invaded, with or withoutdistant metastasis, and (4) D3, in which the metastasis progressesdespite intense hormonal therapy. Id. Treatment at this stage mayinvolve hormonal therapy, chemotherapy, and removal of one or bothtestes. Burdette, supra at 151.

[0013] Despite the need for accurate staging of prostate cancer,current staging methodology is limited. The wide variety ofbiological behavior displayed by neoplasms of the prostate hasresulted in considerable difficulty in predicting and assessing thecourse of prostate cancer. Augustus et al., supra at 47. Indeed,despite the fact that most prostate cancer patients have carcinomasthat are of intermediate grade and stage, prognosis for these typesof carcinomas is highly variable. Andrew A Renshaw &Christopher L. Corless, Prognostic Features in the Pathology ofProstate Cancer, in Prostate Cancer: A Multidisciplinary Guide 26(Philip W. Kantoff et al. eds. 1997). Techniques such astransrectal ultrasound, abdominal and pelvic computerizedtomography, and MRI have not been particularly useful in predictinglocal tumor extension. D'Amico, supra at 53 (editors' comment).While the use of serum PSA in combination with the Gleason score iscurrently the most effective method of staging prostate cancer,id., PSA is of limited predictive value, Augustus et al., supra at47; Renshaw et al., supra at 26, and the Gleason score is prone tovariability and error, King, C. R. & Long, J. P., Int'l. J.Cancer 90(6): 326-30 (2000). As such, the current focus of prostatecancer research has been to obtain biomarkers to help better assessthe progression of the disease. Augustus et al., supra at 47;Renshaw et al., supra at 26; Pettaway, C. A., Tech. Urol. 4(1):35-42 (1998).

[0014] Accordingly, there is a great need for more sensitive andaccurate methods for predicting whether a person is likely todevelop prostate cancer, for diagnosing prostate cancer, formonitoring the progression of the disease, for staging the prostatecancer, for determining whether the prostate cancer hasmetastasized and for imaging the prostate cancer. There is also aneed for better treatment of prostate cancer.

Colon Cancer

[0015] Colorectal cancer is the second most common cause of cancerdeath in the United States and the third most prevalent cancer inboth men and women. M. L. Davila & A. D. Davila, Screening forColon and Rectal Cancer, in Colon and Rectal Cancer 47 (Peter S.Edelstein ed., 2000). Colorectal cancer is categorized as adigestive system cancer by the American Cancer Society (ACS) whichalso includes cancers of the esophagus, stomach, small intestine,anus, anal canal, anorectum, liver & intrahepatic bile duct,gallbladder & other biliary, pancreas, and other digestiveorgans. The ACS estimates that there will be about 263,060 newcases of digestive system cancers in 2006 in the United Statesalone. Digestive system cancers will cause an estimated 136,180deaths combined in the United States in 2006. Specifically, The ACSestimates that there will be about 104,950 new cases of coloncancer, 40,340 new cases of rectal cancer and 5,420 new cases ofsmall intestine cancer in the 2005 in the United States alone.Colon, rectal and small intestine cancers will cause an estimated57,360 deaths combined in the United States in 2005. ACS Website:cancer with the extension .org of the world wide web. Nearly allcases of colorectal cancer arise from adenomatous polyps, some ofwhich mature into large polyps, undergo abnormal growth anddevelopment, and ultimately progress into cancer. Davila at 55-56.This progression would appear to take at least 10 years in mostpatients, rendering it a readily treatable form of cancer ifdiagnosed early, when the cancer is localized. Davila at 56; WalterJ. Burdette, Cancer: Etiology, Diagnosis, and Treatment 125(1998).

[0016] Although our understanding of the etiology of colon canceris undergoing continual refinement, extensive research in this areapoints to a combination of factors, including age, hereditary andnonhereditary conditions, and environmental/dietary factors. Age isa key risk factor in the development of colorectal cancer, Davilaat 48, with men and women over 40 years of age become increasinglysusceptible to that cancer, Burdette at 126. Incidence ratesincrease considerably in each subsequent decade of life. Davila at48. A number of hereditary and nonhereditary conditions have alsobeen linked to a heightened risk of developing colorectal cancer,including familial adenomatous polyposis (FAP), hereditarynonpolyposis colorectal cancer (Lynch syndrome or HNPCC), apersonal and/or family history of colorectal cancer or adenomatouspolyps, inflammatory bowel disease, diabetes mellitus, and obesity.Id. at 47; Henry T. Lynch & Jane F. Lynch, HereditaryNonpolyposis Colorectal Cancer (Lynch Syndromes), in Colon andRectal Cancer 67-68 (Peter S. Edelstein ed., 2000).

[0017] Environmental/dietary factors associated with an increasedrisk of colorectal cancer include a high fat diet, intake of highdietary red meat, and sedentary lifestyle. Davila at 47; Reddy, B.S., Prey. Med. 16(4): 460-7 (1987). Conversely,environmental/dietary factors associated with a reduced risk ofcolorectal cancer include a diet high in fiber, folic acid,calcium, and hormone-replacement therapy in post-menopausal women.Davila at 50-55. The effect of antioxidants in reducing the risk ofcolon cancer is unclear. Davila at 53.

[0018] Because colon cancer is highly treatable when detected at anearly, localized stage, screening should be a part of routine carefor all adults starting at age 50, especially those withfirst-degree relatives with colorectal cancer. One major advantageof colorectal cancer screening over its counterparts in other typesof cancer is its ability to not only detect precancerous lesions,but to remove them as well. Davila at 56. The key colorectal cancerscreening tests in use today are fecal occult blood test,sigmoidoscopy, colonoscopy, double-contrast barium enema, and thecarcinoembryonic antigen (CEA) test. Burdette at 125; Davila at56.

[0019] The fecal occult blood test (FOBT) screens for colorectalcancer by detecting the amount of blood in the stool, the premisebeing that neoplastic tissue, particularly malignant tissue, bleedsmore than typical mucosa, with the amount of bleeding increasingwith polyp size and cancer stage. Davila at 56-57. While effectiveat detecting early stage tumors, FOBT is unable to detectadenomatous polyps (premalignant lesions), and, depending on thecontents of the fecal sample, is subject to rendering falsepositives. Davila at 56-59. Sigmoidoscopy and colonoscopy, bycontrast, allow direct visualization of the bowel, and enable oneto detect, biopsy, and remove adenomatous polyps. Davila at 59-60,61. Despite the advantages of these procedures, there areaccompanying downsides: sigmoidoscopy, by definition, is limited tothe sigmoid colon and below, colonoscopy is a relatively expensiveprocedure, and both share the risk of possible bowel perforationand hemorrhaging. Davila at 59-60. Double-contrast barium enema(DCBE) enables detection of lesions better than FOBT, and almost aswell a colonoscopy, but it may be limited in evaluating the windingrectosigmoid region. Davila at 60. The CEA blood test, whichinvolves screening the blood for carcinoembryonic antigen, sharesthe downside of FOBT, in that it is of limited utility in detectingcolorectal cancer at an early stage. Burdette at 125.

[0020] Once colon cancer has been diagnosed, treatment decisionsare typically made in reference to the stage of cancer progression.A number of techniques are employed to stage the cancer (some ofwhich are also used to screen for colon cancer), includingpathologic examination of resected colon, sigmoidoscopy,colonoscopy, and various imaging techniques. AJCC Cancer StagingHandbook 84 (Irvin D. Fleming et al. eds., 5.sup.th ed. 1998);Montgomery, R. C. and Ridge, J. A., Semin. Surg. Oncol. 15(3):143-150 (1998). Moreover, chest films, liver functionality tests,and liver scans are employed to determine the extent of metastasis.Fleming at 84. While computerized tomography and magnetic resonanceimaging are useful in staging colorectal cancer in its laterstages, both have unacceptably low staging accuracy for identifyingearly stages of the disease, due to the difficulty that bothmethods have in (1) revealing the depth of bowel wall tumorinfiltration and (2) diagnosing malignant adenopathy. Thoeni, R.F., Radiol. Clin. N. Am. 35(2): 457-85 (1997). Rather, techniquessuch as transrectal ultrasound (TRUS) are preferred in thiscontext, although this technique is inaccurate with respect todetecting small lymph nodes that may contain metastases. DavidBlumberg & Frank G. Opelka, Neoadjuvant and Adjuvant Therapyfor Adenocarcinoma of the Rectum, in Colon and Rectal Cancer 316(Peter S. Edelstein ed., 2000). Several classification systems havebeen devised to stage the extent of colorectal cancer, includingthe Dukes' system and the more detailed International Union againstCancer-American Joint Committee on Cancer TNM staging system, whichis considered by many in the field to be a more useful stagingsystem. Burdette at 126-27. The TNM system, which is used foreither clinical or pathological staging, is divided into fourstages, each of which evaluates the extent of cancer growth withrespect to primary tumor (T), regional lymph nodes (N), and distantmetastasis (M). Fleming at 84-85. The system focuses on the extentof tumor invasion into the intestinal wall, invasion of adjacentstructures, the number of regional lymph nodes that have beenaffected, and whether distant metastasis has occurred. Fleming at81.

[0021] Stage 0 is characterized by in situ carcinoma (Tis), inwhich the cancer cells are located inside the glandular basementmembrane (intraepithelial) or lamina propria (intramucosal). Inthis stage, the cancer has not spread to the regional lymph nodes(N0), and there is no distant metastasis (M0). In stage I, there isstill no spread of the cancer to the regional lymph nodes and nodistant metastasis, but the tumor has invaded the submucosa (T1) orhas progressed further to invade the muscularis propria (T2). StageII also involves no spread of the cancer to the regional lymphnodes and no distant metastasis, but the tumor has invaded thesubserosa, or the nonperitonealized pericolic or perirectal tissues(T3), or has progressed to invade other organs or structures,and/or has perforated the visceral peritoneum (T4). Stage III ischaracterized by any of the T substages, no distant metastasis, andeither metastasis in 1 to 3 regional lymph nodes (N1) or metastasisin four or more regional lymph nodes (N2). Lastly, stage IVinvolves any of the T or N substages, as well as distantmetastasis. Fleming at 84-85; Burdette at 127.

[0022] Currently, pathological staging of colon cancer ispreferable over clinical staging as pathological staging provides amore accurate prognosis. Pathological staging typically involvesexamination of the resected colon section, along with surgicalexamination of the abdominal cavity. Fleming at 84. Clinicalstaging would be a preferred method of staging were it at least asaccurate as pathological staging, as it does not depend on theinvasive procedures of its counterpart.

[0023] Turning to the treatment of colorectal cancer, surgicalresection results in a cure for roughly 50% of patients.Irradiation is used both preoperatively and postoperatively intreating colorectal cancer. Chemotherapeutic agents, particularly5-fluorouracil, are also powerful weapons in treating colorectalcancer. Other agents include irinotecan and floxuridine, cisplatin,levamisole, methotrexate, interferon-.alpha., and leucovorin.Burdette at 125, 132-33. Nonetheless, thirty to forty percent ofpatients will develop a recurrence of colon cancer followingsurgical resection, which in many patients is the ultimate cause ofdeath. Wayne De Vos, Follow-up After Treatment of Colon Cancer,Colon and Rectal Cancer 225 (Peter S. Edelstein ed., 2000).Accordingly, colon cancer patients must be closely monitored todetermine response to therapy and to detect persistent or recurrentdisease and metastasis.

[0024] The next few paragraphs describe the some of molecular basesof colon cancer. In the case of FAP, the tumor suppressor gene APC(adenomatous polyposis coli), chromosomally located at 5q21, hasbeen either inactivated or deleted by mutation. Alberts et al.,Molecular Biology of the Cell 1288 (3d ed. 1994). The APC proteinplays a role in a number of functions, including cell adhesion,apoptosis, and repression of the c-myc oncogene. N. R. Hall &R. D. Madoff, Genetics and the Polyp-Cancer Sequence, Colon andRectal Cancer 8 (Peter S. Edelstein, ed., 2000). Of those patientswith colorectal cancer who have normal APC genes, over 65% havesuch mutations in the cancer cells but not in other tissues.Alberts et al., supra at 1288. In the case of HPNCC, patientsmanifest abnormalities in the tumor suppressor gene HNPCC, but onlyabout 15% of tumors contain the mutated gene. Id. A host of othergenes have also been implicated in colorectal cancer, including theK-ras, N-ras, H-ras and c-myc oncogenes, and the tumor suppressorgenes DCC (deleted in colon carcinoma) and p53. Hall & Madoff,supra at 8-9; Alberts et al., supra at 1288.

[0025] Abnormalities in Wg/Wnt signal transduction pathway are alsoassociated with the development of colorectal carcinoma. Taipale,J. and Beachy, P. A. Nature 411: 349-354 (2001). Wnt1 is a secretedprotein gene originally identified within mouse mammary cancers byits insertion into the mouse mammary tumor virus (MMTV) gene. Theprotein is homologous to the wingless (Wg) gene product ofDrosophila, in which it functions as an important factor for thedetermination of dorsal-ventral segmentation and regulates theformation of fly imaginal discs. Wg/Wnt pathway controls cellproliferation, death and differentiation. Taipal (2001). There areat least 13 members in the Wnt family. These proteins have beenfound expressed mainly in the central nervous system (CNS) ofvertebrates as well as other tissues such as mammary and intestine.The Wnt proteins are the ligands for a family of seventransmembrane domain receptors related to the Frizzled gene productin Drosophila. Binding Wnt to Frizzled stimulates the activity ofthe downstream target, Disheveled, which in turn inactivates theglycogen synthetase kinase 3.beta. (GSK3.beta.). Taipal (2001).Usually active GSK3.beta. will form a complex with the adenomatouspolyposis coli (APC) protein and phosphorylate another complexmember, .beta.-catenin. Once phosphorylated, .beta.-catenin isdirected to degradation through the ubiquitin pathway. WhenGSK3.beta. or APC activity is down regulated, .beta.-catenin isaccumulated in the cytoplasm and binds to the T-cell factor orlymphocyte excitation factor (Tcf/Lef) family of transcriptionalfactors. Binding of .beta.-catenin to Tcf releases thetranscriptional repression and induces gene transcription. Amongthe genes regulated by .beta.-catenin are a transcriptionalrepressor Engrailed, a transforming growth factor-.beta.(TGF-.beta.) family member Decapentaplegic, and the cytokineHedgehog in Drosophila. .beta.-Catenin also involves in regulatingcell adhesion by binding to .alpha.-catenin and E-cadherin. On theother hand, binding of .beta.-catenin to these proteins controlsthe cytoplasmic .beta.-catenin level and its complexing with TCF.Taipal (2001). Growth factor stimulation and activation of c-src orv-src also regulate .beta.-catenin level by phosphorylation of.alpha.-catenin and its related protein, p120.sup.cas. Whenphosphorylated, these proteins decrease their binding to E-cadherinand .beta.-catenin resulting in the accumulation of cytoplasmic.beta.-catenin. Reynolds, A. B. et al. Mol. Cell Biol. 14:8333-8342 (1994). In colon cancer, c-src enzymatic activity hasbeen shown increased to the level of v-src. Alternation ofcomponents in the Wg/Wnt pathway promotes colorectal carcinomadevelopment. The best known modifications are to the APC gene.Nicola S et al. Hum. Mol. Genet 10:721-733 (2001). This germlinemutation causes the appearance of hundreds to thousands ofadenomatous polyps in the large bowel. It is the gene defect thataccounts for the autosomally dominantly inherited

[0026] FAP and related syndromes. The molecular alternations thatoccur in this pathway largely involve deletions of alleles oftumor-suppressor genes, such as APC, p53 and Deleted in ColorectalCancer (DCC), combined with mutational activation ofproto-oncogenes, especially c-Ki-ras. Aoki, T. et al. Human Mutat.3: 342-346 (1994). All of these lead to genomic instability incolorectal cancers.

[0027] Another source of genomic instability in colorectal canceris the defect of DNA mismatch repair (MMR) genes. Human homologuesof the bacterial mutHLS complex (hMSH2, hMLHI, hPMS1, hPMS2 andhMSH6), which is involved in the DNA mismatch repair in bacteria,have been shown to cause the HNPCC (about 70-90% HNPCC) whenmutated. Modrich, P. and Lahue, R. Ann Rev. Biochem. 65: 101-133(1996); and Peltomaki, P. Hum. Mol. Genet 10: 735-740 (2001). Theinactivation of these proteins leads to the accumulation ofmutations and causes genetic instability that represents errors inthe accurate replication of the repetitive mono-, di-, tri- andtetra-nucleotide repeats, which are scattered throughout the genome(microsatellite regions). Jass, J. R. et al. J. GastroenterolHepatol 17: 17-26 (2002). Like in the classic FAP, mutationalactivation of c-Ki-ras is also required for the promotion of MSI inthe alternative HNPCC. Mutations in other proteins such as thetumor suppressor protein phosphatase PTEN (Zhou, X. P. et al. Hum.Mol. Genet 11: 445-450 (2002)), BAX (Buttler, L. M. Aus. N. Z. J.Surg. 69: 88-94 (1999)), Caspase-5 (Planck, M. Cancer GenetCytogenet. 134: 46-54 (2002)), TGF.beta.-RII (Fallik, D. et al.Gastroenterol Clin Biol. 24: 917-22 (2000)) and IGFII-R(Giovannucci E. J. Nutr. 131: 3109S-20S (2001)) have also beenfound in some colorectal tumors possibly as the cause of MMRdefect.

[0028] Some tyrosine kinases have been shown up-regulated incolorectal tumor tissues or cell lines like HT29. Skoudy, A. et al.Biochem J. 317 (Pt 1): 279-84 (1996). Focal adhesion kinase (FAK)and its up-stream kinase c-src and c-yes in colonic epithelia cellsmay play an important role in the promotion of colorectal cancersthrough the extracellular matrix (ECM) and integrin-mediatedsignaling pathways. Jessup, J. M. et al., The molecular biology ofcolorectal carcinoma, in: The Molecular Basis of Human Cancer,251-268 (Coleman W. B. and Tsongalis G. J. Eds. 2002). Theformation of c-src/FAK complexes may coordinately deregulate VEGFexpression and apoptosis inhibition. Recent evidences suggest thata specific signal-transduction pathway for cell survival thatimplicates integrin engagement leads to FAK activation and thusactivates PI-3 kinase and akt. In turn, akt phosphorylates BAD andblocks apoptosis in epithelial cells. The activation of c-src incolon cancer may induce VEGF expression through the hypoxiapathway. Other genes that may be implicated in colorectal cancerinclude Cox enzymes (Ota, S. et al. Aliment Pharmacol. Ther. 16(Suppl 2): 102-106 (2002)), estrogen (al-Azzawi, F. and Wahab, M.Climacteric 5: 3-14 (2002)), peroxisome proliferator-activatedreceptor-.gamma. (PPAR-.gamma.) (Gelman, L. et al. Cell Mol. LifeSci. 55: 932-943 (1999)), IGF-I

[0029] (Giovannucci (2001)), thymine DNA glycosylase (TDG)(Hardeland, U. et al. Prog. Nucleic Acid Res. Mol. Biol. 68:235-253 (2001)) and EGF (Mendelsohn, J. Endocrine-Related Cancer 8:3-9 (2001)).

[0030] Gene deletion and mutation are not the only causes fordevelopment of colorectal cancers. Epigenetic silencing by DNAmethylation also accounts for the lost of function of colorectalcancer suppressor genes. A strong association between MSI and CpGisland methylation has been well characterized in sporadiccolorectal cancers with high MSI but not in those of hereditaryorigin. In one experiment, DNA methylation of MLH1, CDKN2A, MGMT,THBS1, RARB, APC, and p14ARF genes has been shown in 80%, 55%, 23%,23%, 58%, 35%, and 50% of 40 sporadic colorectal cancers with highMSI respectively. Yamamoto, H. et al. Genes Chromosomes Cancer 33:322-325 (2002); and Kim, K. M. et al. Oncogene. 12;21(35): 5441-9(2002). Carcinogen metabolism enzymes such as GST, NAT, CYP andMTHFR are also associated with an increased or decreased colorectalcancer risk. Pistorius, S. et al. Kongressbd Dtsch Ges Chir Kongr118: 820-824 (2001); and Potter, J. D. J. Natl. Cancer Inst. 91:916-932 (1999).

[0031] From the foregoing, it is clear that procedures used fordetecting, diagnosing, monitoring, staging, prognosticating, andpreventing the recurrence of colorectal cancer are of criticalimportance to the outcome of the patient. Moreover, currentprocedures, while helpful in each of these analyses, are limited bytheir specificity, sensitivity, invasiveness, and/or their cost. Assuch, highly specific and sensitive procedures that would operateby way of detecting novel markers in cells, tissues, or bodilyfluids, with minimal invasiveness and at a reasonable cost, wouldbe highly desirable.

[0032] Accordingly, there is a great need for more sensitive andaccurate methods for predicting whether a person is likely todevelop colorectal cancer, for diagnosing colorectal cancer, formonitoring the progression of the disease, for staging thecolorectal cancer, for determining whether the colorectal cancerhas metastasized, and for imaging the colorectal cancer. Followingaccurate diagnosis, there is also a need for less invasive and moreeffective treatment of colorectal cancer.

[0033] In addition to prostate and digestive tract cancers, thereis great need for improved methods and compounds for the detection,diagnosis, prognosis, imaging, determination of metastases andmetastatic potential, monitoring, staging and treatment of lung,liver, pancreatic, bone and esophagus cancers.

Angiogenesis in Cancer

[0034] Growth and metastasis of solid tumors are also dependent onangiogenesis. Folkman, J., 1986, Cancer Research, 46, 467-473;Folkman, J., 1989, Journal of the National Cancer Institute, 82,4-6. It has been shown, for example, that tumors which enlarge togreater than 2 mm must obtain their own blood supply and do so byinducing the growth of new capillary blood vessels. Once these newblood vessels become embedded in the tumor, they provide a meansfor tumor cells to enter the circulation and metastasize to distantsites such as liver, lung or bone. Weidner, N., et al., 1991, TheNew England Journal of Medicine, 324(1), 1-8.

[0035] Angiogenesis, defined as the growth or sprouting of newblood vessels from existing vessels, is a complex process thatprimarily occurs during embryonic development. The process isdistinct from vasculogenesis, in that the new endothelial cellslining the vessel arise from proliferation of existing cells,rather than differentiating from stem cells. The process isinvasive and dependent upon proteolysis of the extracellular matrix(ECM), migration of new endothelial cells, and synthesis of newmatrix components. Angiogenesis occurs during embryogenicdevelopment of the circulatory system; however, in adult humans,angiogenesis only occurs as a response to a pathological condition(except during the reproductive cycle in women).

[0036] Under normal physiological conditions in adults,angiogenesis takes place only in very restricted situations such ashair growth and wounding healing. Auerbach, W. and Auerbach, R.,1994, Pharmacol Ther. 63(3):265-3 11; Ribatti et al.,1991,Haematologica 76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-4.Angiogenesis progresses by a stimulus which results in theformation of a migrating column of endothelial cells. Proteolyticactivity is focused at the advancing tip of this "vascular sprout",which breaks down the ECM sufficiently to permit the column ofcells to infiltrate and migrate. Behind the advancing front, theendothelial cells differentiate and begin to adhere to each other,thus forming a new basement membrane. The cells then ceaseproliferation and finally define a lumen for the new arteriole orcapillary.

[0037] Unregulated angiogenesis has gradually been recognized to beresponsible for a wide range of disorders, including, but notlimited to, cancer, cardiovascular disease, rheumatoid arthritis,psoriasis and diabetic retinopathy. Folkman, 1995, Nat Med1(1):27-31; Isner, 1999, Circulation 99(13): 1653-5; Koch, 1998,Arthritis Rheum 41(6):951-62; Walsh, 1999, Rheumatology (Oxford)38(2):103-12; Ware and Simons, 1997, Nat Med 3(2): 158-64.

[0038] Of particular interest is the observation that angiogenesisis required by solid tumors for their growth and metastases.Folkman, 1986 supra; Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6;Folkman, 1992, Semin Cancer Biol 3(2):65-71; Zetter, 1998, Annu RevMed 49:407-24. A tumor usually begins as a single aberrant cellwhich can proliferate only to a size of a few cubic millimeters dueto the distance from available capillary beds, and it can stay`dormant` without further growth and dissemination for a longperiod of time. Some tumor cells then switch to the angiogenicphenotype to activate endothelial cells, which proliferate andmature into new capillary blood vessels. These newly formed bloodvessels not only allow for continued growth of the primary tumor,but also for the dissemination and recolonization of metastatictumor cells. The precise mechanisms that control the angiogenicswitch is not well understood, but it is believed thatneovascularization of tumor mass results from the net balance of amultitude of angiogenesis stimulators and inhibitors Folkman, 1995,supra.

[0039] One of the most potent angiogenesis inhibitors is endostatinidentified by O'Reilly and Folkman. O'Reilly et al., 1997, Cell88(2):277-85; O'Reilly et al., 1994, Cell 79(2):3 15-28. Itsdiscovery was based on the phenomenon that certain primary tumorscan inhibit the growth of distant metastases. O'Reilly and Folkmanhypothesized that a primary tumor initiates angiogenesis bygenerating angiogenic stimulators in excess of inhibitors. However,angiogenic inhibitors, by virtue of their longer half life in thecirculation, reach the site of a secondary tumor in excess of thestimulators. The net result is the growth of primary tumor andinhibition of secondary tumor. Endostatin is one of a growing listof such angiogenesis inhibitors produced by primary tumors. It is aproteolytic fragment of a larger protein: endostatin is a 20 kDafragment of collagen XVIII (amino acid H1132-K1315 in murinecollagen XVIII). Endostatin has been shown to specifically inhibitendothelial cell proliferation in vitro and block angiogenesis invivo. More importantly, administration of endostatin totumor-bearing mice leads to significant tumor regression, and notoxicity or drug resistance has been observed even after multipletreatment cycles. Boehm et al., 1997, Nature 390(6658):404-407. Thefact that endostatin targets genetically stable endothelial cellsand inhibits a variety of solid tumors makes it a very attractivecandidate for anticancer therapy. Fidler and Ellis, 1994, Cell79(2):185-8; Gastl et al., 1997, Oncology 54(3):177-84; Hinsberghet al., 1999, Ann Oncol 10 Suppl 4:60-3. In addition, angiogenesisinhibitors have been shown to be more effective when combined withradiation and chemotherapeutic agents. Klement, 2000, J. ClinInvest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86,Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998,Nature 394(6690):287-91.

[0040] As discussed above, each of the methods for diagnosing andstaging prostate, colon, lung or pancreas cancer is limited by thetechnology employed. Accordingly, there is need for sensitivemolecular and cellular markers for the detection of prostate,colon, lung or pancreas cancer. There is a need for molecularmarkers for the accurate staging, including clinical andpathological staging, of prostate, colon, lung or pancreas cancersto optimize treatment methods. In addition, there is a need forsensitive molecular and cellular markers to monitor the progress ofcancer treatments, including markers that can detect recurrence ofprostate, colon, lung or pancreas cancers following remission.

[0041] The present invention provides alternative methods oftreating prostate, colon, lung or pancreas cancer that overcome thelimitations of conventional therapeutic methods as well as offeradditional advantages that will be apparent from the detaileddescription below.

SUMMARY OF THE INVENTION

[0042] This invention is directed to an isolated Pro115 antibodythat binds to Pro115 on a mammalian cell. The invention is furtherdirected to an isolated Pro115 antibody that internalizes uponbinding to Pro115 on a mammalian cell. The antibody may be amonoclonal antibody. Alternatively, the antibody is an antibodyfragment or a chimeric or a humanized antibody. The monoclonalantibody may be produced by a hybridoma selected from the group ofhybridomas deposited under American Type Culture Collection on 19May 2006 comprising Pro115.B7.1 and Pro115.B34.1.

[0043] The antibody may compete for binding to the same epitope asthe epitope bound by the monoclonal antibody produced by ahybridoma selected from the group of hybridomas deposited under theAmerican Type Culture Collection on 19 May 2006 comprisingPro115.B7.1 and Pro115.B34.1.

[0044] The invention is also directed to conjugated antibodies.They may be conjugated to a growth inhibitory agent or a cytotoxicagent. The cytotoxic agent may be selected from the groupconsisting of toxins, antibiotics, radioactive isotopes andnucleolytic enzymes and toxins. Examples of toxins include, but arenot limited to, maytansin, maytansinoids, saporin, gelonin, ricinor calicheamicin.

[0045] The mammalian cell may be a cancer cell. Preferably, theanti-Pro115 monoclonal antibody that inhibits the growth ofPro115-expressing cancer cells.

[0046] The antibody may be produced in bacteria. Alternatively, theantibody may be a humanized form of an anti-Pro115 antibodyproduced by a hybridoma selected from the group of hybridomasdeposited with the ATCC on 19 May 2006 comprising Pro115.B7.1 andPro115.B34.1.

[0047] Preferably, the cancer is selected from the group consistingof prostate, colon, lung and pancreas cancer. The invention is alsodirected to a method of producing the antibodies comprisingculturing an appropriate cell and recovering the antibody from thecell culture.

[0048] The invention is also directed to compositions comprisingthe antibodies and a carrier. The antibody may be conjugated to acytotoxic agent. The cytotoxic agent may be a radioactive isotopeor other chemotherapeutic agent.

[0049] The invention is also directed to a method of killing anPro115-expressing cancer cell, comprising contacting the cancercell with the antibodies of this invention, thereby killing thecancer cell. The cancer cell may be selected from the groupconsisting of prostate, colon, lung and pancreas cancer cell.

[0050] The prostate, colon, lung or pancreas may be metastaticcancer. The invention is also directed to a method of alleviatingan Pro115-expressing cancer in a mammal, comprising administering atherapeutically effective amount of the antibodies to themammal.

[0051] In addition, the invention is directed to an article ofmanufacture comprising a container and a composition containedtherein, wherein the composition comprises an antibody as describedherein. The article of manufacture may also comprise an additionalcomponent, e.g., a package insert indicating that the compositioncan be used to treat prostate, colon, lung or pancreas cancer.

BRIEF DESCRIPTION OF THE FIGURES

[0052] FIG. 1 shows an alignment between human Pro115, mousePro115, and other human members of the TMPRSS family.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

[0053] Human "Pro115" as used herein, refers to a serine proteaseprotein of 492 amino acids that is expressed on the cell surface,whose nucleotide and amino acid sequences are disclosed wholly orin part in e.g., U.S. Pat. No. 6,043,033, U.S. Pat. No. 6,350,448and US publication 20020119531 as HUPAP; U.S. Pat. No. 7,037,667 as20P1F12/TMPRSS2 gene (also designated as 20P1F12-GTC1); WO 00/12758A1 as sequence gi.beta.2507612|gb|U75329.1|HSU75329 Human serineprotease mRNA, complete CDS; U.S. Pat. No. 6,902,892 as Pro115; andWO 00/00605 A1 as TMPRSS2; the disclosures of which are herebyexpressly incorporated by reference. Amino acids 106-492 of Pro115are expressed on the cell surface. Additionally, Pro115 is shedfrom the cell surface and is present in intercellular matrix andbodily fluids. Amino acids 106-492, or 225-492 (autocleavedprotease domain) of Pro115 are present in intercellular matrix andbodily fluids. Pro115 as used herein includes allelic variants andconservative substitution mutants of the protein which have Pro115biological activity. Specifically, Pro115 includes SNP variantsdescribed herein.

[0054] Pro115 is related to the family of transmembrane serineproteases and is identified in the RefSeq database as accessionsNM.sub.--005656 and NP.sub.--005647 (accessible at ncbi with theextension .nlm.nih.gov of the world wide web) and titled "Homosapiens transmembrane protease, serine 2 (TMPRSS2)". Other synonymsfor Pro115 include: PRSS10 and human epitheliasin. The refseqdatabase includes the following summary of Pro115: [0055] This geneencodes a protein that belongs to the serine protease family. Theencoded protein contains a type II transmembrane domain, a receptorclass A domain, a scavenger receptor cysteine-rich domain and aprotease domain. Serine proteases are known to be involved in manyphysiological and pathological processes. This gene wasdemonstrated to be up-regulated by androgenic hormones in prostatecancer cells and down-regulated in androgen-independent prostatecancer tissue. The protease domain of this protein is thought to becleaved and secreted into cell media after autocleavage. Thebiological function of this gene is unknown. Several publicationshave described the identification, characterization, associationwith carcinomas, and clinical development of Pro115 as a moleculartarget for cancer therapy, diagnosis and vaccination including thefollowing which are hereby incorporated by reference in theirentirety.

TABLE-US-00001 [0055] Tu JJ, et al. Gene fusions between TMPRSS2and ETS family genes in prostate cancer: frequency and transcriptvariant analysis by RT-PCR and FISH on paraffin-embedded tissues.Mod Pathol. 2007 Jul 13; [Epub ahead of print] Yoshimoto M et al.Microdeletion and concurrent translocation associated with acomplex TMPRSS2: ERG prostate cancer gene fusion. Genes ChromosomesCancer. 2007 Sep; 46(9): 861-3. Moore-Scott BA, et al.Identification of molecular markers that are expressed in discreteanterior- posterior domains of the endoderm from the gastrula stageto mid-gestation. Dev Dyn. 2007 Jul; 236(7): 1997-2003. Perner S,et al. TMPRSS2-ERG fusion prostate cancer: an early molecular eventassociated with invasion. Am J Surg Pathol. 2007 Jun; 31(6): 882-8.Mostaghel EA, et al. Intraprostatic androgens andandrogen-regulated gene expression persist after testosteronesuppression: therapeutic implications for castration-resistantprostate cancer. Cancer Res. 2007 May 15; 67(10): 5033-41. Cai C,et al. ETV1 Is a Novel Androgen Receptor-Regulated Gene ThatMediates Prostate Cancer Cell Invasion. Mol Endocrinol. 2007 May15; [Epub ahead of print] Schroder FH. Re: Recurrent fusion ofTMPRSS2 and ETS transcription factor genes in prostate cancer. EurUrol. 2007 May; 51(5): 1443-4. Billis A. Morphological features ofTMPRSS2: ERG fusion prostate cancer. Int Braz J Urol. 2007 Mar-Apr;33(2): 282-3. Billis A. Are there morphologic correlates ofprostate cancer associated with TMPRSS2-ERG molecularabnormalities? Int Braz J Urol. 2007 Mar-Apr; 33(2): 281-2.Saramaki O, Visakorpi T. Chromosomal aberrations in prostatecancer. Front Biosci. 2007 May 1; 12: 3287-301. Perner S, et al.[TMPRSS2-ETS gene fusion in prostate cancer.] Urologe A. 2007 Jul;46(7): 754-760. Teixeira MR. Recurrent fusion oncogenes incarcinomas. Crit Rev Oncog. 2006 Dec; 12(3-4): 257-71. Review.Nelson WG. Prostate cancer prevention. Curr Opin Urol. 2007 May;17(3): 157-67. 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Frequency of theTMPRSS2: ERG gene fusion is increased in moderate to poorlydifferentiated prostate cancers. J Clin Pathol. 2007 Jan 26; [Epubahead of print] Demichelis F, et al. TMPRSS2: ERG gene fusionassociated with lethal prostate cancer in a watchful waitingcohort. Oncogene. 2007 Jul 5; 26(31): 4596-9. Epub 2007 Jan 22.Macaluso M, Giordano A. TMPRSS2: ERG gene fusion a new geneticmarker for prostate cancer progression. Cancer Biol Ther. 2007 Jan;6(1): 46-7. Nami RK, et al. Expression of TMPRSS2: ERG gene fusionin prostate cancer cells is an important prognostic factor forcancer progression. Cancer Biol Ther. 2007 Jan; 6(1): 40-5. HermansKG, et al. TMPRSS2: ERG fusion by translocation or interstitialdeletion is highly relevant in androgen-dependent prostate cancer,but is bypassed in late-stage androgen receptor-negative prostatecancer. Cancer Res. 2006 Nov 15; 66(22): 10658-63. Iljin K, et al.TMPRSS2 fusions with oncogenic ETS factors in prostate cancerinvolve unbalanced genomic rearrangements and are associated withHDAC1 and epigenetic reprogramming. Cancer Res. 2006 Nov 1; 66(21):10242-6. Laxman B, et al. Noninvasive detection of TMPRSS2: ERGfusion transcripts in the urine of men with prostate cancer.Neoplasia. 2006 Oct; 8(10): 885-8. Clark J, et al. Diversity ofTMPRSS2-ERG fusion transcripts in the human prostate. Oncogene.2007 Apr 19; 26(18): 2667-73. Epub 2006 Oct 16. Cerveira N, et al.TMPRSS2-ERG gene fusion causing ERG overexpression precedeschromosome copy number changes in prostate carcinomas and pairedHGPIN lesions. Neoplasia. 2006 Oct; 8(10): 826-32. Kantoff P.Prevention, complementary therapies, and new scientificdevelopments in the field of prostate cancer. Rev Urol. 2006; 8Suppl 2: S9-S14. Rubin MA, Chinnaiyan AM. Bioinformatics approachleads to the discovery of the TMPRSS2: ETS gene fusion in prostatecancer. Lab Invest. 2006 Nov; 86(11): 1099-102. Epub 2006 Sep 18.Jia L, et al. Locus-wide chromatin remodeling and enhanced androgenreceptor-mediated transcription in recurrent prostate tumor cells.Mol Cell Biol. 2006 Oct; 26(19): 7331-41. Bottcher E, et al.Proteolytic activation of influenza viruses by serine proteasesTMPRSS2 and HAT from human airway epithelium. J Virol. 2006 Oct;80(19): 9896-8. Wang J, et al. Expression of Variant TMPRSS2/ERGFusion Messenger RNAs Is Associated with Aggressive ProstateCancer. Cancer Res. 2006 Sep 1; 66(17): 8347-51. Perner S, et al.TMPRSS2: ERG Fusion-Associated Deletions Provide Insight into theHeterogeneity of Prostate Cancer. Cancer Res. 2006 Sep 1; 66(17):8337-41. Liu W, et al. Comprehensive assessment of DNA copy numberalterations in human prostate cancers using Affymetrix 100K SNPmapping array. Genes Chromosomes Cancer. 2006 Nov; 45(11): 1018-32.Yoshimoto M, Jos et al. Three-Color FISH Analysis of TMPRSS2/ERGFusions in Prostate Cancer Indicates That Genomic Microdeletion ofChromosome 21 Is Associated with Rearrangement. Neoplasia. 2006Jun; 8(6): 465-9. Tomlins SA, et al. TMPRSS2: ETV4 gene fusionsdefine a third molecular subtype of prostate cancer. Cancer Res.2006 Apr 1; 66(7): 3396-400. Soller MJ, et al. Confirmation of thehigh frequency of the TMPRSS2/ERG fusion gene in prostate cancer.Genes Chromosomes Cancer. 2006 Jul; 45(7): 717-9. Ahlers CM, FiggWD. ETS-TMPRSS2 fusion gene products in prostate cancer. CancerBiol Ther. 2006 Mar; 5(3): 254-5. Epub 2006 Mar 13. Kim TS, et al.Phenotypic analysis of mice lacking the Tmprss2-encoded protease.Mol Cell Biol. 2006 Feb; 26(3): 965-75. Chen Y, et al. Elevatedexpression and potential roles of human Sp5, a member of Sptranscription factor family, in human cancers. Biochem Biophys ResCommun. 2006 Feb 17; 340(3): 758-66. Epub 2005 Dec 20. Tomlins SA,et al. Recurrent fusion of TMPRSS2 and ETS transcription factorgenes in prostate cancer. Science. 2005 Oct 28; 310(5748): 644-8.Schalken JA, et al. Molecular prostate cancer pathology: currentissues and achievements. Scand J Urol Nephrol Suppl. 2005 May;(216): 82-93. O'Leary DA, et al. Tissue-specific overexpression ofthe HSA21 gene GABPalpha: implications for DS. Biochim BiophysActa. 2004 Dec 24; 1739(1): 81-7. Wilson S, et al. Themembrane-anchored serine protease, TMPRSS2, activates PAR-2 inprostate cancer cells. Biochem J. 2005 Jun 15; 388(Pt 3): 967-72.Hessels D, et al. Applicability of biomarkers in the earlydiagnosis of prostate cancer. Expert Rev Mol Diagn. 2004 Jul; 4(4):513-26. Lubieniecka JM, et al. Met160Val polymorphism in theTRMPSS2 gene and risk of prostate cancer in a population-basedcase-control study. Prostate. 2004 Jun 1; 59(4): 357-9. Hartel A,et al. 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Spinesin/TMPRSS5, a novel transmembrane serine protease, clonedfrom human spinal cord. J Biol Chem. 2002 Mar 1; 277(9): 6806-12.Epub 2001 Dec 12. Davisson MT, et al. Evolutionary breakpoints onhuman chromosome 21. Genomics. 2001 Nov; 78(1-2): 99-106. Teng DH,et al. Mutation analyses of 268 candidate genes in human tumor celllines. Genomics. 2001 Jun 15; 74(3): 352-64. Pletcher MT, et al.Use of comparative physical and sequence mapping to annotate mousechromosome 16 and human chromosome 21. Genomics. 2001 May 15;74(1): 45-54. Jacquinet E, et al. Cloning and characterization ofthe cDNA and gene for human epitheliasin. Eur J Biochem. 2001 May;268(9): 2687-99. Afar DE, et al. Catalytic cleavage of theandrogen-regulated TMPRSS2 protease results in its secretion byprostate and prostate cancer epithelia. Cancer Res. 2001 Feb 15;61(4): 1686-92. Vaarala MH, et al. Expression of transmembraneserine protease TMPRSS2 in mouse and human tissues. J Pathol. 2001Jan; 193(1): 134-40. Jacquinet E, et al. Cloning, genomicorganization, chromosomal assignment and expression of a novelmosaic serine proteinase: epitheliasin. FEBS Lett. 2000 Feb 18;468(1): 93-100. Lin B, et al. Prostate-localized andandrogen-regulated expression of the membrane-bound serine proteaseTMPRSS2. Cancer Res. 1999 Sep 1; 59(17): 4180-4. Hildmann T, et al.A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2based on high-throughput nonisotopic library screenings. GenomeRes. 1999 Apr; 9(4): 360-72. Paoloni-Giacobino A, et al. Cloning ofthe TMPRSS2 gene, which encodes a novel serine protease withtransmembrane, LDLRA, and SRCR domains and maps to 21q22.3.Genomics. 1997 Sep 15; 44(3): 309-20. Erratum in: Genomics 2001Sep; 77(1-2): 114.

[0056] As described in the publications above, Pro115 is membraneprotease that is differentially expressed in prostate, colon andother cancers versus normal tissues. Pro115 biological activityincludes protease activity, protease-activated receptor 2 (PAR-2)activation, sodium channel regulation, fusion protein production,regulation or promotion of carcinogenesis, tumorigenesis, celldifferentiation, migration, vascular morphogenesis orangiogenesis.

[0057] Expression of Pro115, and the increased protease activity,has protective effects for cells (e.g. cancer cells) and confers asurvival benefit. Autocleavage and secretion of the active proteasedomain into the surrounding matrix of a tumor promotes growth,migration and metastases of Pro115 expressing tumors. Inhibition ofPro115 activity prevents tumor growth and spread.

[0058] It has also been shown that due to mutations and genomicrearrangements Pro115 is mutated or part of fusion genes inprostate cancer.

[0059] Recently, several publications have described gene fusionsbetween TMPRSS2 (Pro115) and transcription factors of the ETSfamily, commonly ERG and ETV1. These gene fusions are due togenomic rearrangement or intronic deletion and have been describedas markers of cancer prognosis, progression, differentiation,invasion and morphology. Pro115 fusion proteins produced by thesegene fusions are alternative embodiments of Pro115. Specifically,any fusion protein containing a portion of the Pro115 protein (e.g.TMPRSS2-ERG, TMPRSS2-ETV1) is encompassed by the herein descriptionof Pro115 and antibodies thereto.

[0060] Taken together, the differential expression in cancer,protease activity, and role in cellular processes make Pro115 apromising target for diagnosis and immunotherapy of prostate andother tumor types. Anti-Pro115 antibodies are useful in diagnosticor therapeutic applications alone or in combination with moleculesagainst other TMPRSS family members, serine proteases and growthfactors and their receptors (e.g. VEGF, EGFR).

[0061] The antibodies of the instant invention, those describedpreviously and herein, specifically bind Pro115 and havedemonstrated characteristics which make them ideal therapeuticcandidates for modulating Pro115 functions including proteaseactivity, protease-activated receptor 2 (PAR-2) activation, sodiumchannel regulation, fusion protein production, regulation orpromotion of carcinogenesis, tumorigenesis, cell differentiation,migration, vascular morphogenesis or angiogenesis. Furthermore, theantibodies of the instant invention are useful as therapeuticagents for those suffering from prostate, colon, lung or pancreascancers. The antibodies may have therapeutic effect by killingPro115 expressing cancer cells, inhibiting growth of Pro115expressing tumors, shrinking Pro115 expressing tumors, extendingsurvival time of individuals with Pro115 expressing tumors,reducing metastases of Pro115 expressing tumors, inducing immuneresponse against Pro115 expressing tumors or reducing angiogenesisor vascularization of Pro115 expressing tumors.

[0062] The term "antibody" (Ab) as used herein includes monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments, as long as theyexhibit the desired biological activity. The term "immunoglobulin"(Ig) is used interchangeably with "antibody" herein.

[0063] An "isolated antibody" is one which has been identified andseparated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials which would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. Preferably, the antibodywill be purified (1) to greater than 95% by weight of antibody asdetermined by the Lowry method, and most preferably more than 99%by weight, (2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or non-reducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibodyin situ within recombinant cells since at least one component ofthe antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least onepurification step.

[0064] The basic 4-chain antibody unit is a heterotetramericglycoprotein composed of two identical light (L) chains and twoidentical heavy (H) chains (an IgM antibody consists of 5 of thebasic heterotetramer unit along with an additional polypeptidecalled J chain, and therefore contain 10 antigen binding sites,while secreted IgA antibodies can polymerize to form polyvalentassemblages comprising 2-5 of the basic 4-chain units along with Jchain). In the case of IgGs, the 4-chain unit is generally about150,000 daltons. Each L chain is linked to an H chain by onecovalent disulfide bond, while the two H chains are linked to eachother by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variabledomain (VH) followed by three constant domains (CH) for each of the.alpha. and .gamma. chains and four CH domains for [L and Fisotypes. Each 6 L chain has at the N-terminus, a variable domain(VL) followed by a constant domain (CL) at its other end.

[0065] The VL is aligned with the VH and the CL is aligned with thefirst constant domain of the heavy chain (CHI).

[0066] Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains.The pairing of a VH and VL together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Teff and Tristram G. Parsiow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter6.

[0067] The L chain from any vertebrate species can be assigned toone of two clearly distinct types, called kappa and lambda, basedon the amino acid sequences of their constant domains. Depending onthe amino acid sequence of the constant domain of their heavychains (CH), immunoglobulins can be assigned to different classesor isotypes. There are five classes of immunoglobulins: IgA, IgD,IgE, IgG, and IgM, having heavy chains designated .alpha., .delta.,.epsilon., .gamma. and .mu., respectively. They .gamma. and .alpha.classes are further divided into subclasses on the basis ofrelatively minor differences in C.sub.H sequence and function,e.g., humans express the following subclasses: IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2.

[0068] The term "variable" refers to the fact that certain segmentsof the variable domains differ extensively in sequence amongantibodies. The V domain mediates antigen binding and definespecificity of a particular antibody for its particular antigen.However, the variability is not evenly distributed across the1-10-amino acid span of the variable domains. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called "hypervariable regions" that are each9-12 amino acids long. The variable domains of native heavy andlight chains each comprise four FRs, largely adopting a P-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the P-sheetstructure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC).

[0069] The term "hypervariable region" when used herein refers tothe amino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region generally comprises aminoacid residues from a "complementarity determining region" or "CDR"(e.g. around about residues 24-34 (LI), 5056 (L2) and 89-97 (L3) inthe VL, and around about 1-35 (HI), 50-65 (H2) and 95-102 (113) inthe VH; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes ofHealth, Bethesda, Md. (1991)) and/or those residues from a"hypervariable loop" (e.g. residues 26-32 (LI), 50-52 (L2) and91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and 96-101 (H3)in the VH; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0070] The term "monoclonal antibody" as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. Furthermore, in contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In additionto their specificity, the monoclonal antibodies are advantageous inthat they may be synthesized uncontaminated by other antibodies.The modifier "monoclonal" is not to be construed as requiringproduction of the antibody by any particular method. For example,the monoclonal antibodies useful in the present invention may beprepared by the hybridoma methodology first described by Kohler etal., Nature, 256:495 (1975), or may be made using recombinant DNAmethods in bacterial, eukaryotic animal or plant cells (see, e.g.,U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also beisolated from phage antibody libraries using the techniquesdescribed in Clackson et al., Nature, 352:624-628 (1991) and Markset al., J. Mol. Biol., 222:581-597 (1991), for example. Themonoclonal antibodies herein include "chimeric" antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass,as well as fragments of such antibodies, so long as they exhibitthe desired biological activity (see U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include "primatized"antibodies comprising variable domain antigen-binding sequencesderived from a non-human primate (e.g. Old World Monkey, Ape etc),and human constant region sequences.

[0071] An "intact" antibody is one which comprises anantigen-binding site as well as a CL and at least heavy chainconstant domains, CHI, CH2 and CH3. The constant domains may benative sequence constant domains (e.g. human native sequenceconstant domains) or amino acid sequence variant thereof.Preferably, the intact antibody has one or more effectorfunctions.

[0072] An "antibody fragment" comprises a portion of an intactantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab',F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S.Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called "Fab" fragments, and a residual "Fc" fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variableregion domain of the H chain (VH), and the first constant domain ofone heavy chain (CHI). Each Fab fragment is monovalent with respectto antigen binding, i.e., it has a single antigen-binding site.Pepsin treatment of an antibody yields a single large F(ab')2fragment which roughly corresponds to two disulfide linked Fabfragments having divalent antigen-binding activity and is stillcapable of cross-linking antigen. Fab' fragments differ from Fabfragments by having additional few residues at the carboxy terminusof the CHI domain including one or more cysteines from the antibodyhinge region. Fab'-SH is the designation herein for Fab' in whichthe cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab')2 antibody fragments originally were produced as pairsof 8 Fab' fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

[0073] The Fc fragment comprises the carboxy-terminal portions ofboth H chains held together by disulfides. The effector functionsof antibodies are determined by sequences in the Fc region, whichregion is also the part recognized by Fc receptors (FcR) found oncertain types of cells.

[0074] "Fv" is the minimum antibody fragment which contains acomplete antigen-recognition and -binding site. This fragmentconsists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the foldingof these two domains emanate six hypervariable loops (3 loops eachfrom the H and L chain) that contribute the amino acid residues forantigen binding and confer antigen binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the abilityto recognize and bind antigen, although at a lower affinity thanthe entire binding site.

[0075] "Single-chain Fv" also abbreviated as "sFv" or "scFv" areantibody fragments that comprise the VH and VL antibody domainsconnected into a single polypeptide chain. Preferably, the sFvpolypeptide further comprises a polypeptide linker between the VHand VL domains which enables the sFv to form the desired structurefor antigen binding. For a review of sFv, see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol.

[0076] 113, Rosenburg and Moore eds., Springer-Verlag, N.Y., pp.269-315 (1994); Borrebaeck 1995, infra.

[0077] The term "diabodies" refers to small antibody fragmentsprepared by constructing sFv fragments (see preceding paragraph)with short linkers (about 5-10 residues) between the VH and VLdomains such that inter-chain but not intra-chain pairing of the Vdomains is achieved, resulting in a bivalent fragment, i.e.,fragment having two antigen-binding sites. Bispecific diabodies areheterodimers of two "crossover" sFv fragments in which the VH andVL domains of the two antibodies are present on differentpolypeptide chains. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993). Furthermore, effects of linkersequence alterations in engineering bispecific tandem diabodies aredescribed in Le Gall et al., Protein Eng Des Sel. 17(4):357-66(2004).

[0078] A "native sequence" polypeptide is one which has the sameamino acid sequence as a polypeptide (e.g., antibody) derived fromnature. Such native sequence polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. Thus,a native sequence polypeptide can have the amino acid sequence of anaturally occurring human polypeptide, murine polypeptide, orpolypeptide from any other mammalian species.

[0079] The term "amino acid sequence variant" refers to apolypeptide that has amino acid sequences that differ to someextent from a native sequence polypeptide. Ordinarily, amino acidsequence variants of Pro115 will possess at least about 70%homology with the native sequence Pro115, preferably, at leastabout 80%, more preferably at least about 85%, even more preferablyat least about 90% homology, and most preferably at least 95%. Theamino acid sequence variants can possess substitutions, deletions,insertions and/or alterations due to allelic variation or SingleNucleotide Polymorphisms (SNPs) within the native nucleic acidsequence encoding the amino acid sequence.

[0080] Several definitions of SNPs exist. See, e.g., Brooks, 235Gene 177-86 (1999). As used herein, the term "single nucleotidepolymorphism" or "SNP" includes all single base variants, thusincluding nucleotide insertions and deletions in addition to singlenucleotide substitutions and any resulting amino acid variants dueto codon alteration. There are two types of nucleotidesubstitutions. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. Atransversion is the replacement of a purine for a pyrimidine, orvice versa.

[0081] Numerous methods exist for detecting SNPs within anucleotide sequence. A review of many of these methods can be foundin Landegren et al., 8 Genome Res. 769-76 (1998). For example, aSNP in a genomic sample can be detected by preparing a ReducedComplexity Genome (RCG) from the genomic sample, then analyzing theRCG for the presence or absence of a SNP. See, e.g., WO 00/18960.Multiple SNPs in a population of target polynucleotides in parallelcan be detected using, for example, the methods of WO 00/50869.Other SNP detection methods include the methods of U.S. Pat. Nos.6,297,018 and 6,322,980. Furthermore, SNPs can be detected byrestriction fragment length polymorphism (RFLP) analysis. See,e.g., U.S. Pat. Nos. 5,324,631; 5,645,995. RFLP analysis of SNPs,however, is limited to cases where the SNP either creates ordestroys a restriction enzyme cleavage site. SNPs can also bedetected by direct sequencing of the nucleotide sequence ofinterest. In addition, numerous assays based on hybridization havealso been developed to detect SNPs and mismatch distinction bypolymerases and ligases. Several web sites provide informationabout SNPs including Ensembl (ensembl with the extension .org ofthe world wide web), Sanger Institute (sanger with the extension.ac.uk/genetics/exon/ of the world wide web), National Center forBiotechnology Information (NCBI) (ncbi with the extension .nlm.nih.gov/SNP/ of the world wide web), The SNP Consortium Ltd.(snp.cshl.org). The chromosomal locations for the compositionsdisclosed herein are provided below. In addition, one of ordinaryskill in the art could perform a search against the genome or anyof the databases cited above using BLAST to find the chromosomallocation or locations of SNPs. Another a preferred method to findthe genomic coordinates and associated SNPs would be to use theBLAT tool (genome.ucsc.edu, Kent et al. 2001, The Human GenomeBrowser at UCSC, Genome Research 996-1006 or Kent 2002 BLAT, TheBLAST-Like Alignment Tool Genome Research, 1-9). All web sitesabove were accessed Dec. 3, 2003.

[0082] Preferred amino acid sequence variants of Pro115 aredescribed in the table below. The polynucleotides encoding theamino acids of the present invention were analyzed and singlenucleotide polymorphism (SNP) attributes were identified.Specifically identified were SNPs occurring the coding region ofthe nucleotide, the Alleles of the SNP, the nucleotide ambiguitycode for the SNP, the position in the codon of the SNP if withinthe Open Reading Frame (1, 2, 3 or UTR for untranslated regions),and the SNP type (synonymous or non-synonymous to the proteintranslation). In addition to the attributes above, the SNP rs#IDfor the NCBI SNP database (dbSNP) which is accessible at ncbi withthe extension .nlm.nih.gov/SNP/ of the world wide web is referencedfor each SNP. Additional single nucleotide polymorphism (SNP)information can be accessed at the databases listed above.

[0083] The table below includes the polynucleotide target, dbSNPrs#ID, Nucleic acid residue affected by the SNP (Polynucleotide) inNM.sub.--005656, SNP alleles, Nucleotide ambiguity code, CondonPosition of the SNP if within the ORF (1, 2, 3 or UTR if not withinORF), and the SNP type (synonymous "syn" or non-synonymous"non-syn"), Amino acid residue affected by the SNP (AA Residue) inNP.sub.--005647, and the Alternate amino acid residue.

TABLE-US-00002 Nucleic Amino dbSNP Acid Ambiguity Codon AcidAlternate rs# ID Residue Alleles Code Pos SNP type Residue AminoAcid Pro115 28532009 162 G/A R 2 Non-syn 12 G/E Pro115 3787950 352A/G R 3 Syn 75 T/T Pro115 12329760 605 G/A R 1 Non-syn 160 M/VPro115 17854725 895 T/C Y 3 Syn 256 I/I Pro115 2298659 904 C/T Y 3Syn 259 G/G Pro115 2298658 919 G/A R 3 Syn 264 P/P Pro115 10566021474 G/C S 3 Non-syn 449 K/N

[0084] Variants of Pro115 as described above or in the literatureand antibodies which bind to these variants are part of theinvention described herein.

[0085] The phrase "functional fragment or analog" of an antibody isa compound having qualitative biological activity in common with afull-length antibody. For example, a functional fragment or analogof an anti-IgE antibody is one which can bind to an IgEimmunoglobulin in such a manner so as to prevent or substantiallyreduce the ability of such molecule from having the ability to bindto the high affinity receptor, Fc.epsilon.RI.

[0086] "Homology" is defined as the percentage of residues in theamino acid sequence variant that are identical after aligning thesequences and introducing gaps, if necessary, to achieve themaximum percent homology. Methods and computer programs for thealignment are well known in the art. Sequence similarity may bemeasured by any common sequence analysis algorithm, such as GAP orBESTFIT or other variation Smith-Waterman alignment. See, T. F.Smith and M. S. Waterman, J. Mol. Biol. 147:195-197 (1981) and W.R. Pearson, Genomics 11:635-650 (1991).

[0087] "Humanized" forms of non-human (e.g., rodent) antibodies arechimeric antibodies that contain minimal sequence derived from thenon-human antibody. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues froma hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit or non-human primate having the desiredantibody specificity, affinity, and capability. In some instances,framework region (FR) residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore,humanized antibodies may comprise residues that are not found inthe recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all ofat least one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those ofa non-human immunoglobulin and all or substantially all of the FRsare those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0088] As used herein, an anti-Pro115 antibody that "internalizes"is one that is taken up by (i.e., enters) the cell upon binding toPro115 on a mammalian cell (i.e. cell surface Pro115). Theinternalizing antibody will of course include antibody fragments,human or humanized antibody and antibody conjugate. For therapeuticapplications, internalization in vivo is contemplated. The numberof antibody molecules internalized will be sufficient or adequateto kill an Pro115-expressing cell, especially an Pro115-expressingcancer cell. Depending on the potency of the antibody or antibodyconjugate, in some instances, the uptake of a single antibodymolecule into the cell is sufficient to kill the target cell towhich the antibody binds. For example, certain toxins are highlypotent in killing such that internalization of one molecule of thetoxin conjugated to the antibody is sufficient to kill the tumorcell.

[0089] Whether an anti-Pro115 antibody internalizes upon bindingPro115 on a mammalian cell can be determined by various assaysincluding those described in the experimental examples below. Forexample, to test internalization in vivo, the test antibody islabeled and introduced into an animal known to have Pro115expressed on the surface of certain cells. The antibody can beradiolabeled or labeled with fluorescent or gold particles, forinstance. Animals suitable for this assay include a mammal such asa NCR nude mouse that contains a human Pro115-expressing tumortransplant or xenograft, or a mouse into which cells transfectedwith human Pro115 have been introduced, or a transgenic mouseexpressing the human Pro115 transgene. Appropriate controls includeanimals that did not receive the test antibody or that received anunrelated antibody, and animals that received an antibody toanother antigen on the cells of interest, which antibody is knownto be internalized upon binding to the antigen. The antibody can beadministered to the animal, e.g., by intravenous injection. Atsuitable time intervals, tissue sections of the animal can beprepared using known methods or as described in the experimentalexamples below, and analyzed by light microscopy or electronmicroscopy, for internalization as well as the location of theinternalized antibody in the cell. For internalization in vitro,the cells can be incubated in tissue culture dishes in the presenceor absence of the relevant antibodies added to the culture mediaand processed for microscopic analysis at desired time points. Thepresence of an internalized, labeled antibody in the cells can bedirectly visualized by microscopy or by autoradiography ifradiolabeled antibody is used. Alternatively, in a quantitativebiochemical assay, a population of cells comprisingPro115-expressing cells are contacted in vitro or in vivo with aradiolabeled test antibody and the cells (if contacted in vivo,cells are then isolated after a suitable amount of time) aretreated with a protease or subjected to an acid wash to removeuninternalized antibody on the cell surface. The cells are groundup and the amount of protease resistant, radioactive counts perminute (cpm) associated with each batch of cells is measured bypassing the homogenate through a scintillation counter. Based onthe known specific activity of the radiolabeled antibody, thenumber of antibody molecules internalized per cell can be deducedfrom the scintillation counts of the ground-up cells. Cells are"contacted" with antibody in vitro preferably in solution form suchas by adding the cells to the cell culture media in the culturedish or flask and mixing the antibody well with the media to ensureuniform exposure of the cells to the antibody. Instead of adding tothe culture media, the cells can be contacted with the testantibody in an isotonic solution such as PBS in a test tube for thedesired time period. In vivo, the cells are contacted with antibodyby any suitable method of administering the test antibody such asthe methods of administration described below when administered toa patient.

[0090] The faster the rate of internalization of the antibody uponbinding to the Pro115-expressing cell in vivo, the faster thedesired killing or growth inhibitory effect on the targetPro115-expressing cell can be achieved, e.g., by a cytotoxicimmunoconjugate. Preferably, the kinetics of internalization of theanti-Pro115 antibodies are such that they favor rapid killing ofthe Pro115-expressing target cell. Therefore, it is desirable thatthe anti-Pro115 antibody exhibit a rapid rate of internalizationpreferably, within 24 hours from administration of the antibody invivo, more preferably within about 12 hours, even more preferablywithin about 30 minutes to 1 hour, and most preferably, withinabout 30 minutes. The present invention provides antibodies thatinternalize as fast as about 15 minutes from the time ofintroducing the anti-Pro115 antibody in vivo. The antibody willpreferably be internalized into the cell within a few hours uponbinding to Pro115 on the cell surface, preferably within 1 hour,even more preferably within 15-30 minutes.

[0091] To determine if a test antibody can compete for binding tothe same epitope as the epitope bound by the anti-Pro115 antibodiesof the present invention including the antibodies produced by thehybridomas deposited with the ATCC, a cross-blocking assay e.g., acompetitive ELISA assay can be performed. In an exemplarycompetitive ELISA assay, Pro115-coated wells of a microtiter plate,or Pro115-coated sepharose beads, are pre-incubated with or withoutcandidate competing antibody and then a biotin-labeled anti-Pro115antibody of the invention is added. The amount of labeledanti-Pro115 antibody bound to the Pro115 antigen in the wells or onthe beads is measured using avidin-peroxidase conjugate andappropriate substrate.

[0092] Alternatively, the anti-Pro115 antibody can be labeled,e.g., with a radioactive or fluorescent label or some otherdetectable and measurable label. The amount of labeled anti-Pro115antibody that binds to the antigen will have an inverse correlationto the ability of the candidate competing antibody (test antibody)to compete for binding to the same epitope on the antigen, i.e.,the greater the affinity of the test antibody for the same epitope,the less labeled anti-Pro115 antibody will be bound to theantigen-coated wells. A candidate competing antibody is consideredan antibody that binds substantially to the same epitope or thatcompetes for binding to the same epitope as an anti-Pro115 antibodyof the invention if the candidate competing antibody can blockbinding of the anti-Pro115 antibody by at least 20%, preferably byat least 20-50%, even more preferably, by at least 50% as comparedto a control performed in parallel in the absence of the candidatecompeting antibody (but may be in the presence of a knownnoncompeting antibody). It will be understood that variations ofthis assay can be performed to arrive at the same quantitativevalue.

[0093] An antibody having a "biological characteristic" of adesignated antibody, such as any of the monoclonal antibodiesPro115.A1, Pro115.A2, Pro115.A3, Pro115.A4, Pro115.A5, Pro115.A6,Pro115.A7, Pro115.A8, Pro115.A9, Pro115.A10, Pro115.A11,Pro115.A12, Pro115.A13, Pro115.A14, Pro115.A15, Pro115.A16,Pro115.A17, Pro115.A18, Pro115.A19, Pro115.A20, Pro115A21,Pro115.A22, Pro115.A23, Pro115.A24, Pro115.A25, Pro115.A101.1,Pro115.A102.1, Pro115.A103.1, Pro115.A104.1, Pro115.A106.1,Pro115.A107.1, Pro115.A108.1, Pro115.B1, Pro115.B2, Pro115.B3,Pro115.B4, Pro115.B5, Pro115.B6, Pro115.B7, Pro115.B8, Pro115.B9,Pro115.B10, Pro115.B11, Pro115.B12, Pro115.B13, Pro115.B14,Pro115.B15, Pro115.B16, Pro115.B17, Pro115.B18, Pro115.B19, Pro115.B20, Pro115.B21, Pro115.B22, Pro115.B23, Pro115.B24,Pro115.B25, Pro115.B26, Pro115.B27, Pro115.B28, Pro115.B29,Pro115.B30, Pro115.B31, Pro115.B32, Pro115.B33, Pro115.B34,Pro115.B35, Pro115.B36, Pro115.B37, Pro115.B38, Pro115.B39,Pro115.B40, Pro115.B41, Pro115.B42, Pro115.B43, Pro115.B44,Pro115.B45, Pro115.B46, Pro115.B47, Pro115.B48, Pro115.B49,Pro115.B50, Pro115.B51, Pro115.B52, Pro115.B53, Pro115.B54,Pro115.B55, Pro115.B56, Pro115.B57, Pro115.B58, Pro115.B59,Pro115.B60, Pro115.B61, Pro115.B62, Pro115.B63, Pro115.B64,Pro115.B65, Pro115.B66, Pro115.B67, Pro115.B68, Pro115.B69,Pro115.D1, Pro 115.D2, Pro 115.D3, Pro115.D4, Pro115.D5, Pro115.D6,Pro115.D7, Pro115.D8, Pro115.D9, Pro115.D10, Pro115.D11,Pro115.D12, Pro115.D13, Pro115.F2 and Pro115.F3, is one whichpossesses one or more of the biological characteristics of thatantibody which distinguish it from other antibodies that bind tothe same antigen, Pro115.A1, Pro115.A2, Pro115.A3, Pro115.A4,Pro115.A5, Pro115.A6, Pro115.A7, Pro115.A8, Pro115.A9, Pro115.A10,Pro115.A11, Pro115.A12, Pro115.A13, Pro115.A14, Pro115.A15,Pro115.A16, Pro115.A17, Pro115.A18, Pro115.A19, Pro115.A20,Pro115.A21, Pro115.A22, Pro115.A23, Pro115.A24, Pro115.A25,Pro115.A101.1, Pro115.A102.1, Pro115.A103.1, Pro115.A104.1,Pro115.A106.1, Pro115.A107.1, Pro115.A108.1, Pro115.B1, Pro115.B2,Pro115.B3, Pro115.B4, Pro115.B5, Pro115.B6, Pro115.B7, Pro115.B8,Pro115.B9, Pro115.B10, Pro115.B11, Pro115.B12, Pro115.B13,Pro115.B14, Pro115.B15, Pro115.B16, Pro115.B17, Pro115.B18,Pro115.B19, Pro115.B20, Pro115.B21, Pro115.B22, Pro115.B23,Pro115.B24, Pro115.B25, Pro115.B26, Pro115.B27, Pro115.B28,Pro115.B29, Pro115.B30, Pro115.B31, Pro115.B32, Pro115.B33,Pro115.B34, Pro115.B35, Pro115.B36, Pro115.B37, Pro115.B38,Pro115.B39, Pro115.B40, Pro115.B41, Pro115.B42, Pro115.B43,Pro115.B44, Pro115.B45, Pro115.B46, Pro115.B47, Pro115.B48,Pro115.B49, Pro115.B50, Pro115.B51, Pro115.B52, Pro115.B53,Pro115.B54, Pro115.B55, Pro115.B56, Pro115.B57, Pro115.B58,Pro115.B59, Pro115.B60, Pro115.B61, Pro115.B62, Pro115.B63,Pro115.B64, Pro115.B65, Pro115.B66, Pro115.B67, Pro115.B68,Pro115.B69, Pro115.D1, Pro115.D2, Pro115.D3, Pro115.D4, Pro115.D5,Pro115.D6, Pro115.D7, Pro115.D8, Pro115.D9, Pro115.D10, Pro115.D11,Pro115.D12, Pro115.D13, Pro115.F2 and Pro115.F3 will bind the sameepitope as that bound by Pro115.A1, Pro 115.A2, Pro115.A3,Pro115.A4, Pro115.A5, Pro115.A6, Pro115.A7, Pro115.A8, Pro115.A9,Pro115.A10, Pro115.A11, Pro115.A12, Pro115.A13, Pro115.A14,Pro115.A15, Pro115.A16, Pro115.A17, Pro115.A18, Pro115.A19,Pro115.A20, Pro115.A21, Pro115.A22, Pro115.A23, Pro115.A24,Pro115.A25, Pro115.A101.1, Pro115.A102.1, Pro115.A103.1,Pro115.A104.1, Pro115.A106.1, Pro115.A107.1, Pro115.A108.1,Pro115.B1, Pro115.B2, Pro115.B3, Pro115.B4, Pro115.B5, Pro115.B6,Pro115.B7, Pro115.B8, Pro115.B9, Pro115.B10, Pro115.B11,Pro115.B12, Pro115.B13, Pro115.B14, Pro115.B15, Pro115.B16,Pro115.B17, Pro115.B18, Pro115.B19, Pro115.B20, Pro115.B21,Pro115.B22, Pro115.B23, Pro115.B24, Pro115.B25, Pro115.B26,Pro115.B27, Pro115.B28, Pro115.B29, Pro115.B30, Pro115.B31,Pro115.B32, Pro115.B33, Pro115.B34, Pro115.B35, Pro115.B36,Pro115.B37, Pro115.B38, Pro115.B39, Pro115.B40, Pro115.B41,Pro115.B42, Pro115.B43, Pro115.B44, Pro115.B45, Pro115.B46,Pro115.B47, Pro115.B48, Pro115.B49, Pro115.B50, Pro115.B51,Pro115.B52, Pro115.B53, Pro115.B54, Pro115.B55, Pro115.B56,Pro115.B57, Pro115.B58, Pro115.B59, Pro115.B60, Pro115.B61,Pro115.B62, Pro115.B63, Pro115.B64, Pro115.B65, Pro115.B66,Pro115.B67, Pro115.B68, Pro115.B69, Pro115.D1, Pro115.D2,Pro115.D3, Pro115.D4, Pro115.D5, Pro115.D6, Pro115.D7, Pro115.D8,Pro115.D9, Pro115.D10, Pro115.D11, Pro115.D12, Pro115.D13,Pro115.F2 and Pro115.F3 (e.g. which competes for binding or blocksbinding of monoclonal antibody Pro115.A1, Pro115.A2, Pro115.A3,Pro115.A4, Pro115.A5, Pro115.A6, Pro115.A7, Pro115.A8, Pro115.A9,Pro115.A10, Pro115.A11, Pro115.A12, Pro115.A13, Pro115.A14,Pro115.A15, Pro115.A16, Pro115.A17, Pro115.A18, Pro115.A19,Pro115.A20, Pro115.A21, Pro115.A22, Pro115.A23, Pro115.A24,Pro115.A25, Pro115.A101.1, Pro115.A102.1, Pro115.A103.1,Pro115.A104.1, Pro115.A106.1, Pro115.A107.1, Pro115.A108.1,Pro115.B1, Pro115.B2, Pro115.B3, Pro115.B4, Pro115.B5, Pro115.B6,Pro115.B7, Pro115.B8, Pro115.B9, Pro115.B10, Pro115.B11,Pro115.B12, Pro115.B13, Pro115.B14, Pro115.B15, Pro115.B16,Pro115.B17, Pro115.B18, Pro115.B19, Pro115.B20, Pro115.B21,Pro115.B22, Pro115.B23, Pro115.B24, Pro115.B25, Pro115.B26,Pro115.B27, Pro115.B28, Pro115.B29, Pro115.B30, Pro115.B31,Pro115.B32, Pro115.B33, Pro115.B34, Pro115.B35, Pro115.B36,Pro115.B37, Pro115.B38, Pro115.B39, Pro115.B40, Pro115.B41,Pro115.B42, Pro115.B43, Pro115.B44, Pro115.B45, Pro115.B46,Pro115.B47, Pro115.B48, Pro115.B49, Pro115.B50, Pro115.B51,Pro115.B52, Pro115.B53, Pro115.B54, Pro115.B55, Pro115.B56,Pro115.B57, Pro115.B58, Pro115.B59, Pro115.B60, Pro115.B61,Pro115.B62, Pro115.B63, Pro115.B64, Pro115.B65, Pro115.B66,Pro115.B67, Pro115.B68, Pro115.B69, Pro115.D1, Pro115.D2,Pro115.D3, Pro115.D4, Pro115.D5, Pro115.D6, Pro115.D7, Pro115.D8,Pro115.D9, Pro115.D10, Pro115.D11, Pro115.D12, Pro115.D13,Pro115.F2 and Pro115.F3), be able to target an Pro115-expressingtumor in vivo and may internalize upon binding to Pro115 on amammalian cell in vivo. Likewise, an antibody with the biologicalcharacteristic of the Pro115.A1, Pro115.A2, Pro115.A3, Pro115.A4,Pro115.A5, Pro115.A6, Pro115.A7, Pro115.A8, Pro115.A9, Pro115.A10,Pro115.A11, Pro115.A12, Pro115.A13, Pro115.A14, Pro115.A15,Pro115.A16, Pro115.A17, Pro115.A18, Pro115.A19, Pro115.A20,Pro115.A21, Pro115.A22, Pro115.A23, Pro115.A24, Pro115.A25,Pro115.A101.1, Pro115.A102.1, Pro115.A103.1, Pro115.A104.1,Pro115.A106.1, Pro115.A107.1, Pro115.A108.1, Pro115.B1, Pro115.B2,Pro115.B3, Pro115.B4, Pro115.B5, Pro115.B6, Pro115.B7, Pro115.B8,Pro115.B9, Pro115.B10, Pro115.B11, Pro115.B12, Pro115.B13,Pro115.B14, Pro115.B15, Pro115.B16, Pro115.B17, Pro115.B18,Pro115.B19, Pro115.B20, Pro115.B21, Pro115.B22, Pro115.B23,Pro115.B24, Pro115.B25, Pro115.B26, Pro115.B27, Pro115.B28,Pro115.B29, Pro115.B30, Pro115.B31, Pro115.B32, Pro115.B33,Pro115.B34, Pro115.B35, Pro115.B36, Pro115.B37, Pro115.B38,Pro115.B39, Pro115.B40, Pro115.B41, Pro115.B42, Pro115.B43,Pro115.B44, Pro115.B45, Pro115.B46, Pro115.B47, Pro115.B48,Pro115.B49, Pro115.B50, Pro115.B51, Pro115.B52, Pro115.B53,Pro115.B54, Pro115.B55, Pro115.B56, Pro115.B57, Pro115.B58,Pro115.B59, Pro115.B60, Pro115.B61, Pro115.B62, Pro115.B63,Pro115.B64, Pro115.B65, Pro115.B66, Pro115.B67, Pro115.B68,Pro115.B69, Pro115.D1, Pro115.D2, Pro115.D3, Pro 115.D4, Pro115.D5,Pro115.D6, Pro115.D7, Pro115.D8, Pro115.D9, Pro115.D10, Pro115.D11,Pro115.D12, Pro115.D13, Pro115.F2 and Pro115.F3 antibody will havethe same epitope binding, targeting, internalizing, tumor growthinhibitory and cytotoxic properties of the antibody.

[0094] The term "antagonist" antibody is used in the broadestsense, and includes an antibody that partially or fully blocks,inhibits, or neutralizes a biological activity of a native Pro115protein disclosed herein. Methods for identifying antagonists of anPro115 polypeptide may comprise contacting an Pro115 polypeptide ora cell expressing Pro115 on the cell surface, with a candidateantagonist antibody and measuring a detectable change in one ormore biological activities normally associated with the Pro115polypeptide.

[0095] The term `agonistic" antibody is used in the broadest sense,and includes an antibody the partially or fully promotes,activates, or increases biological activity of Pro115.Additionally, an agonistic antibody may mimic an Pro115 bindingpartner (e.g. receptor or ligand) wherein binding of the Pro115antibody has substantially the same effect on biologic activity ofPro115 as binding of the binding partner. Methods for identifyingagonists of an Pro115 polypeptide may comprise contacting an Pro115polypeptide or a cell expressing Pro115 on the cell surface, with acandidate agonistic antibody and measuring a detectable change inone or more biological activities normally associated with thePro115 polypeptide.

[0096] An "antibody that inhibits the growth of tumor cellsexpressing Pro115" or a "growth inhibitory" antibody is one whichbinds to and results in measurable growth inhibition of cancercells expressing or overexpressing Pro115. Preferred growthinhibitory anti-Pro115 antibodies inhibit growth ofPro115-expressing tumor cells (e.g., prostate, colon, lung orpancreas cancer cells) by greater than 20%, preferably from about20% to about 50%, and even more preferably, by greater than 50%(e.g. from about 50% to about 100%) as compared to the appropriatecontrol, the control typically being tumor cells not treated withthe antibody being tested. Growth inhibition can be measured at anantibody concentration of about 0.1 to 30 pg/ml or about 0.5 nM to200 nM in cell culture, where the growth inhibition is determined1-10 days after exposure of the tumor cells to the antibody. Growthinhibition of tumor cells in vivo can be determined in various wayssuch as is described in the Experimental Examples section below.The antibody is growth inhibitory in vivo if administration of theanti-Pro115 antibody at about 1 pg/kg to about 100 mg/kg bodyweight results in reduction in tumor size or tumor cellproliferation within about 5 days to 3 months from the firstadministration of the antibody, preferably within about 5 to 30days.

[0097] An antibody which "induces apoptosis" is one which inducesprogrammed cell death as determined by binding of annexin V,fragmentation of DNA, cell shrinkage, dilation of endoplasmicreticulum, cell fragmentation, and/or formation of membranevesicles (called apoptotic bodies). The cell is usually one whichoverexpresses Pro115. Preferably the cell is a tumor cell, e.g. anovarian, colon, prostate, or lung cell. Various methods areavailable for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation canbe measured by annexin binding; DNA fragmentation can be evaluatedthrough DNA laddering; and nuclear/chromatin condensation alongwith DNA fragmentation can be evaluated by any increase inhypodiploid cells. Preferably, the antibody which induces apoptosisis one which results in about 2 to 50 fold, preferably about 5 to50 fold, and most preferably about 10 to 50 fold, induction ofannexin binding relative to untreated cells in an annexin bindingassay.

[0098] Antibody "effector functions" refer to those biologicalactivities attributable to the Fc region (a native sequence Fcregion or amino acid sequence variant Fc region) of an antibody,and vary with the antibody isotype. Examples of antibody effectorfunctions include: C1q binding and complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); complement dependent cytotoxicity (CDC);phagocytosis; down regulation of cell surface receptors (e.g. Bcell receptor); and B cell activation.

[0099] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"refers to a form of cytotoxicity in which secreted Ig bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g. NaturalKiller (NK) cells, neutrophils, and macrophages) enable thesecytotoxic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently kill the target cell with cytotoxins.The antibodies "arm" the cytotoxic cells and are absolutelyrequired for such killing. The primary cells for mediating ADCC, NKcells, express Fc.gamma.RIII only, whereas monocytes expressFc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetchand Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such asthat described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such asthat disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0100] "Fc receptor" or "FcR" describes a receptor that binds tothe Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds anIgG antibody (a gamma receptor) and includes receptors of theFc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, includingallelic variants and alternatively spliced forms of thesereceptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an"activating receptor") and Fc.gamma.RIIB (an "inhibitingreceptor"), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof Activating receptorFc.gamma.RIIA contains an immunoreceptor tyrosine-based activationmotif (ITAM) in its cytoplasmic domain. Inhibiting receptorFc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibitionmotif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel etal., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.Clin. Med. 126.330-41 (1995). Other FcRs, including those to beidentified in the future, are encompassed by the term "FcR" herein.The term also includes the neonatal receptor, FcRn, which isresponsible for the transfer, of maternal IgGs to the fetus (Guyeret al., J.

[0101] Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)).

[0102] "Human effector cells" are leukocytes which express one ormore FcRs and perform effector functions. Preferably, the cellsexpress at least Fc.gamma.RIII and perform ADCC effector function.Examples of human leukocytes which mediate ADCC include peripheralblood mononuclear cells (PBMC), natural killer (NK) cells,monocytes, cytotoxic T cells and neutrophils; with PBMCs and NKcells being preferred. The effector cells may be isolated from anative source, e.g. from blood.

[0103] "Complement dependent cytotoxicity" or "CDC" refers to thelysis of a target cell in the presence of complement. Activation ofthe classical complement pathway is initiated by the binding of thefirst component of the complement system (C1q) to antibodies (ofthe appropriate subclass) which are bound to their cognate antigen.To assess complement activation, a CDC assay, e.g. as described inGazzano-Santoro et al., J. Immunol. Methods 202:163 (1996) may beperformed.

[0104] The terms "cancer" and "cancerous" refer to or describe thephysiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include, but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancersinclude squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,liver cancer, bladder cancer, cancer of the urinary tract,hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma, anal carcinoma, penile carcinoma,melanoma, multiple myeloma and B-cell lymphoma, brain, as well ashead and neck cancer, and associated metastases.

[0105] A "Pro115-expressing cell" is a cell which expressesendogenous or transfected Pro115 on the cell surface. A"Pro115-expressing cancer" is a cancer comprising cells that havePro115 protein present on the cell surface. A "Pro115-expressingcancer" produces sufficient levels of Pro115 on the surface ofcells thereof, such that an anti-Pro115 antibody can bind theretoand have a therapeutic effect with respect to the cancer. A cancerwhich "overexpresses" Pro115 is one which has higher levels ofPro115 at the cell surface thereof, compared to a noncancerous cellof the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation. Pro115overexpression may be determined in a diagnostic or prognosticassay by evaluating increased levels of the Pro115 protein presenton the surface of a cell (e.g. via an immunohistochemistry assay;FACS analysis). Alternatively, or additionally, one may measurelevels of Pro115-encoding nucleic acid or mRNA in the cell, e.g.via fluorescent in situ hybridization; (FISH; see W098/45479published October, 1998), Southern blotting, Northern blotting, orpolymerase chain reaction (PCR) techniques, such as real timequantitative PCR (RT-PCR). One may also study Pro115 overexpressionby measuring shed antigen in a biological fluid such as serum,e.g., using antibody-based assays (see also, e.g., U.S. Pat. No.4,933,294 issued Jun. 12, 1990; W091/05264 published Apr. 18, 1991;U.S. Pat. 5,401,638 issued Mar. 28, 1995; and Sias et al. J.Immunol. Methods 132: 73-80 (1990)). Aside from the above assays,various in vivo assays are available to the skilled practitioner.For example, one may expose cells within the body of the patient toan antibody which is optionally labeled with a detectable label,e.g. a radioactive isotope, and binding of the antibody to cells inthe patient can be evaluated, e.g. by external scanning forradioactivity or by analyzing a biopsy taken from a patientpreviously exposed to the antibody. A Pro115-expressing cancerincludes prostate, colon, lung or pancreas cancer.

[0106] A "mammal" for purposes of treating a cancer or alleviatingthe symptoms of cancer, refers to any mammal, including-humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the mammal is human.

[0107] "Treating" or "treatment" or "alleviation" refers to boththerapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) the targetedpathologic condition or disorder. Those in need of treatmentinclude those already with the disorder as well as those prone tohave the disorder or those in whom the disorder is to be prevented.A subject or mammal is successfully "treated" for anPro115-expressing cancer if, after receiving a therapeutic amountof an anti-Pro115 antibody according to the methods of the presentinvention, the patient shows observable and/or measurable reductionin or absence of one or more of the following: reduction in thenumber of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent andpreferably stop) of cancer cell infiltration into peripheral organsincluding the spread of cancer into soft tissue and bone;inhibition (i.e., slow to some extent and preferably stop) of tumormetastasis; inhibition, to some extent, of tumor growth; and/orrelief to some extent, one or more of the symptoms associated withthe specific cancer; reduced morbidity and mortality, andimprovement in quality of life issues. To the extent theanti-Pro115 antibody may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Reduction of thesesigns or symptoms may also be felt by the patient.

[0108] The above parameters for assessing successful treatment andimprovement in the disease are readily measurable by routineprocedures familiar to a physician. For cancer therapy, efficacycan be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

[0109] The term "therapeutically effective amount" refers to anamount of an antibody or a drug effective to "treat" a disease ordisorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the drug may reduce the numberof cancer cells; reduce the tumor size; inhibit (i.e., slow to someextent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent andpreferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. See preceding definition of "treating".To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic.

[0110] "Chronic" administration refers to administration of theagent(s) in a continuous mode as opposed to an acute mode, so as tomaintain the initial therapeutic effect (activity) for an extendedperiod of time.

[0111] "Intermittent" administration is treatment that is notconsecutively done without interruption, but rather is cyclic innature.

[0112] Administration "in combination with" one or more furthertherapeutic agents includes simultaneous (concurrent) andconsecutive administration in any order. "Carriers" as used hereininclude pharmaceutically acceptable carriers, excipients, orstabilizers which are nontoxic to the cell or mammal being exposedthereto at the dosages and concentrations employed.

[0113] Often the physiologically acceptable carrier is an aqueouspH buffered solution. Examples of physiologically acceptablecarriers include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; salt-forming counterions such as sodium;and/or nonionic surfactants such as TWEEN.TM., polyethylene glycol(PEG), and PLURONICS.TM..

[0114] The term "cytotoxic agent" as used herein refers to asubstance that inhibits or prevents the function of cells and/orcauses destruction of cells. The term is intended to includeradioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,and radioactive isotopes of Lu), chemotherapeutic agents e.g.methotrexate, adriamicin, vinca alkaloids (vincristine,vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,chlorambucil, daunorubicin or other intercalating agents, enzymesand fragments thereof such as nucleolytic enzymes, antibiotics, andtoxins such as small molecule toxins or enzymatically active toxinsof bacterial, fungal, plant or animal origin, including fragmentsand/or variants thereof, e.g., gelonin, ricin, saporin, and thevarious antitumor or anticancer agents disclosed below. Othercytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

[0115] A "growth inhibitory agent" when used herein refers to acompound or composition which inhibits growth of a cell, especiallya Pro 115-expressing cancer cell, either in vitro or in vivo. Thus,the growth inhibitory agent may be one which significantly reducesthe percentage of Pro115-expressing cells in S phase. Examples ofgrowth inhibitory agents include agents that block cell cycleprogression (at a place other than S phase), such as agents thatinduce GI arrest and M-phase arrest. Classical M-phase blockersinclude the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest GIalso spill over into S-phase arrest, for example, DNA alkylatingagents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,cisplatin, methotrexate, 5-fluorouracil, and ara-C. Furtherinformation can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycleregulation, oncogenes, and antineoplastic drugs" by Murakami et al.(WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes(paclitaxel and docetaxel) are anticancer drugs both derived fromthe yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),derived from the European yew, is a semisynthetic analogue ofpaclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel anddocetaxel promote the assembly of microtubules from tubulin dimersand stabilize microtubules by preventing depolymerization, whichresults in the inhibition of mitosis in cells.

[0116] "Label" as used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to theantibody so as to generate a "labeled" antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable.

[0117] The term "epitope tagged" used herein refers to a chimericpolypeptide comprising an anti-Pro115 antibody polypeptide fused toa "tag polypeptide". The tag polypeptide has enough residues toprovide an epitope against which an antibody can be made, yet isshort enough such that it does not interfere with activity of theIg polypeptide to which it is fused. The tag polypeptide is alsopreferably fairly unique so that the antibody does notsubstantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues andusually between about 8 and 50 amino acid residues (preferably,between about 10 and 20 amino acid residues).

[0118] A "small molecule" is defined herein to have a molecularweight below about 500 Daltons.

[0119] The term "package insert" is used to refer to instructionscustomarily included in commercial packages of therapeuticproducts, that contain information about the indications, usage,dosage, administration, contraindications and/or warningsconcerning the use of such therapeutic products.

[0120] An "isolated nucleic acid molecule" is a nucleic acidmolecule, e.g., an RNA, DNA, or a mixed polymer, which issubstantially separated from other genome DNA sequences as well asproteins or complexes such as ribosomes and polymerases, whichnaturally accompany a native sequence. The term embraces a nucleicacid molecule which has been removed from its naturally occurringenvironment, and includes recombinant or cloned DNA isolates andchemically synthesized analogues or analogues biologicallysynthesized by heterologous systems. A substantially pure nucleicacid molecule includes isolated forms of the nucleic acidmolecule.

[0121] "Vector" includes shuttle and expression vectors andincludes, e.g., a plasmid, cosmid, or phagemid. Typically, aplasmid construct will also include an origin of replication (e.g.,the ColEl origin of replication) and a selectable marker (e.g.,ampicillin or tetracycline resistance), for replication andselection, respectively, of the plasmids in bacteria. An"expression vector" refers to a vector that contains the necessarycontrol sequences or regulatory elements for expression of theantibodies including antibody fragment of the invention, inprokaryotic, e.g., bacterial, or eukaryotic cells. Suitable vectorsare disclosed below.

[0122] The cell that produces an anti-Pro115 antibody of theinvention will include the parent hybridoma cell e.g., thehybridomas that are deposited with the ATCC, as well as bacterialand eukaryotic host cells into which nucleic acid encoding theantibodies have been introduced. Suitable host cells are disclosedbelow.

[0123] RNA interference refers to the process of sequence-specificpost transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to as posttranscriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of posttranscriptional gene silencing is thought to be an evolutionarilyconserved cellular defense mechanism used to prevent the expressionof foreign genes which is commonly shared by diverse flora andphyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double stranded RNAs (dsRNA) derived from viralinfection or the random integration of transposon elements into ahost genome via a cellular response that specifically destroyshomologous single stranded RNA or viral genomic RNA. The presenceof dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears tobe different from the interferon response that results from dsRNAmediated activation of protein kinase PKR and 2',5'-oligoadenylatesynthetase resulting in non-specific cleavage of mRNA byribonuclease L.

[0124] The presence of long dsRNAs in cells stimulates the activityof a ribonuclease III enzyme referred to as dicer. Dicer isinvolved in the processing of the dsRNA into short pieces of dsRNAknown as short interfering RNAs (siRNA) (Berstein et al., 2001,Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21-23 nucleotides in length andcomprise about 19 base pair duplexes. Dicer has also beenimplicated in the excision of 21 and 22 nucleotide small temporalRNAs (stRNA) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of singlestranded RNA having sequence complementary to the antisense strandof the siRNA duplex. Cleavage of the target RNA takes place in themiddle of the region complementary to the antisense strand of thesiRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

[0125] Short interfering RNA mediated RNAi has been studied in avariety of systems. Fire et al., 1998, Nature, 391, 806, were thefirst to observe RNAi in C. Elegans. Wianny and Goetz, 1999, NatureCell Biol., 2, 70, describe RNAi mediated by dsRNA in mouseembryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi inDrosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexesof synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20,6877) has revealed certain requirements for siRNA length,structure, chemical composition, and sequence that are essential tomediate efficient RNAi activity. These studies have shown that 21nucleotide siRNA duplexes are most active when containing twonucleotide 3'-overhangs. Furthermore, complete substitution of oneor both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methylnucleotides abolishes RNAi activity, whereas substitution of the3'-terminal siRNA overhang nucleotides with deoxynucleotides (2'-H)was shown to be tolerated. Single mismatch sequences in the centerof the siRNA duplex were also shown to abolish RNAi activity. Inaddition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5'-end of thesiRNA guide sequence rather than the 3'-end (Elbashir et al., 2001,EMBO J., 20, 6877). Other studies have indicated that a5'-phosphate on the target-complementary strand of a siRNA duplexis required for siRNA activity and that ATP is utilized to maintainthe 5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell,107, 309).

[0126] Studies have shown that replacing the 3'-overhangingsegments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to 4 nucleotides on each end of the siRNAwith deoxyribonucleotides has been reported to be well toleratedwhereas complete substitution with deoxyribonucleotides results inno RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). Inaddition, Elbashir et al., supra, also report that substitution ofsiRNA with 2'-O-methyl nucleotides completely abolishes RNAiactivity. Li et al., International PCT Publication No. WO 00/44914,and Beach et al., International PCT Publication No. WO 01/68836both suggest that siRNA "may include modifications to either thephosphate-sugar back bone or the nucleoside to include at least oneof a nitrogen or sulfur heteroatom", however neither applicationteaches to what extent these modifications are tolerated in siRNAmolecules nor provide any examples of such modified siRNA. Kreutzerand Limmer, Canadian Patent Application No. 2,359,180, alsodescribe certain chemical modifications for use in dsRNA constructsin order to counteract activation of double stranded-RNA-dependentprotein kinase PKR, specifically 2'-amino or 2'-O-methylnucleotides, and nucleotides containing a 2'-O or 4'-C methylenebridge. However, Kreutzer and Limmer similarly fail to show to whatextent these modifications are tolerated in siRNA molecules nor dothey provide any examples of such modified siRNA.

[0127] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, testedcertain chemical modifications targeting the unc-22 gene in C.elegans using long (>25 nt) siRNA transcripts. The authorsdescribe the introduction of thiophosphate residues into thesesiRNA transcripts by incorporating thiophosphate nucleotide analogswith T7 and T3 RNA polymerase and observed that "RNAs with two(phosphorothioate) modified bases also had substantial decreases ineffectiveness as RNAi triggers (data not shown); (phosphorothioate)modification of more than two residues greatly destabilized theRNAs in vitro and we were not able to assay interferenceactivities." Id. at 1081. The authors also tested certainmodifications at the 2'-position of the nucleotide sugar in thelong siRNA transcripts and observed that substitutingdeoxynucleotides for ribonucleotides "produced a substantialdecrease in interference activity", especially in the case ofUridine to Thymidine and/or Cytidine to deoxy-Cytidinesubstitutions. Id. In addition, the authors tested certain basemodifications, including substituting 4-thiouracil, 5-bromouracil,5-iodouracil, 3-(aminoallyl)uracil for uracil, and inosine forguanosine in sense and antisense strands of the siRNA, and foundthat whereas 4-thiouracil and 5-bromouracil were all welltolerated, inosine "produced a substantial decrease in interferenceactivity" when incorporated in either strand. Incorporation of5-iodouracil and 3-(aminoallyl)uracil in the antisense strandresulted in substantial decrease in RNAi activity as well.

[0128] Beach et al., International PCT Publication No. WO 01/68836,describes specific methods for attenuating gene expression usingendogenously derived dsRNA. Tuschl et al., International PCTPublication No. WO 01/75164, describes a Drosophila in vitro RNAisystem and the use of specific siRNA molecules for certainfunctional genomic and certain therapeutic applications; althoughTuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can beused to cure genetic diseases or viral infection due "to the dangerof activating interferon response". Li et al., International PCTPublication No. WO 00/44914, describes the use of specific dsRNAsfor use in attenuating the expression of certain target genes.Zernicka-Goetz et al., International PCT Publication No. WO01/36646, describes certain methods for inhibiting the expressionof particular genes in mammalian cells using certain dsRNAmolecules. Fire et al., International PCT Publication No. WO99/32619, describes particular methods for introducing certaindsRNA molecules into cells for use in inhibiting gene expression.Plaetinck et al., International PCT Publication No. WO 00/01846,describes certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describes the identification ofspecific genes involved in dsRNA mediated RNAi. DeschampsDepaillette et al., International PCT Publication No. WO 99/07409,describes specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Driscoll et al.,International PCT Publication No. WO 01/49844, describes specificDNA constructs for use in facilitating gene silencing in targetedorganisms. Parrish et al., 2000, Molecular Cell, 6, 1977-1087,describes specific chemically modified siRNA constructs targetingthe unc-22 gene of C. elegans. Tuschl et al., International PCTPublication No. WO 02/44321, describe certain synthetic siRNAconstructs.

Compositions and Methods of the Invention

[0129] The invention provides anti-Pro115 antibodies. Preferably,the anti-Pro115 antibodies internalize upon binding to cell surfacePro115 on a mammalian cell. The anti-Pro115 antibodies may alsodestroy or lead to the destruction of tumor cells bearingPro115.

[0130] It was not apparent that Pro115 wasinternalization-competent. In addition the ability of an antibodyto internalize depends on several factors including the affinity,avidity, and isotype of the antibody, and the epitope that itbinds. We have demonstrated herein that the cell surface Pro115 isinternalization competent upon binding by the anti-Pro115antibodies of the invention. Additionally, it was demonstrated thatthe anti-Pro115 antibodies of the present invention canspecifically target Pro115-expressing tumor cells. These tumortargeting, internalization and growth inhibitory properties of theanti-Pro115 antibodies make these antibodies very suitable fortherapeutic uses, e.g., in the treatment of various cancersincluding prostate, colon, lung or pancreas cancer. Internalizationof the anti-Pro115 antibody is preferred, e.g., if the antibody orantibody conjugate has an intracellular site of action and if thecytotoxic agent conjugated to the antibody does not readily crossthe plasma membrane (e.g., the toxin calicheamicin).Internalization is not necessary if the antibodies or the agentconjugated to the antibodies do not have intracellular sites ofaction, e.g., if the antibody can kill the tumor cell by ADCC orsome other mechanism.

[0131] The anti-Pro115 antibodies of the invention also havevarious non-therapeutic applications. The anti-Pro115 antibodies ofthe present invention can be useful for diagnosis and staging ofPro115-expressing cancers (e.g., in radioimaging). They may be usedalone or in combination with other ovarian cancer markers,including, but not limited to, CA125, HE4 and mesothelin. Theantibodies are also useful for purification or immunoprecipitationof Pro115 from cells, for detection and quantitation of Pro115 invitro, e.g. in an ELISA or a Western blot, to kill and eliminatePro115-expressing cells from a population of mixed cells as a stepin the purification of other cells. The internalizing anti-Pro115antibodies of the invention can be in the different formsencompassed by the definition of "antibody" herein. Thus, theantibodies include full length or intact antibody, antibodyfragments, native sequence antibody or amino acid variants,humanized, chimeric or fusion antibodies, immunoconjugates, andfunctional fragments thereof. In fusion antibodies, an antibodysequence is fused to a heterologous polypeptide sequence. Theantibodies can be modified in the Fc region to provide desiredeffector functions. As discussed in more detail in the sectionsbelow, with the appropriate Fc regions, the naked antibody bound onthe cell surface can induce cytotoxicity, e.g., viaantibody-dependent cellular cytotoxicity (ADCC) or by recruitingcomplement in complement dependent cytotoxicity, or some othermechanism. Alternatively, where it is desirable to eliminate orreduce effector function, so as to minimize side effects ortherapeutic complications, certain other Fc regions may beused.

[0132] The antibody may compete for binding, or binds substantiallyto, the same epitope bound by the antibodies of the invention.Antibodies having the biological characteristics of the presentanti-Pro115 antibodies of the invention are also contemplated,e.g., an anti-Pro115 antibody which has the biologicalcharacteristics of a monoclonal antibody produced by the hybridomasdeposited with the ATCC on 19 May 2006 comprising Pro115.B7.1 andPro115.B34.1, specifically including the in vivo tumor targeting,internalization and any cell proliferation inhibition or cytotoxiccharacteristics. Specifically provided are anti-Pro115 antibodiesthat bind to an epitope present in amino acids 1-10, 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,120-130, 130-140, 140-150, 150-160, 160-170, 170-180,180-190,190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260,260-270, 270-280, 280-290, 290-300, 300-310, 310,-320, 320-330,330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400,400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470,470-480, 480-490, 490-492 or 1-15, 10-25, 20-35, 30-45, 40-55,50-65, 60-75, 70-85, 80-95, 90-105, 100-115, 110-125, 120-135,130-145, 140-155, 150-165, 160-175, 170-185, 180-195, 190-205,200-215, 210-225, 220-235, 230-245, 240-255, 250-265, 260-275,270-285, 280-295, 290-305, 300-315, 310-325, 320-335, 330-345,340-355, 350-365, 360-375, 370-385, 380-395, 390-405, 400-415,410-425, 420-435, 430-445, 440-455, 450-465, 460-475, 470-485,480-492 or Peptides 1-39 described in detail below of humanPro115.

[0133] Methods of producing the above antibodies are described indetail below.

[0134] The present anti-Pro115 antibodies are useful for treating aPro115-expressing cancer or alleviating one or more symptoms of thecancer in a mammal. Such a cancer includes prostate, colon, lung orpancreas cancer, cancer of the urinary tract, lung cancer, breastcancer, colon cancer, pancreatic cancer, and ovarian cancer, morespecifically, prostate adenocarcinoma, renal cell carcinomas,colorectal adenocarcinomas, lung adenocarcinomas, lung squamouscell carcinomas, and pleural mesothelioma. The cancers encompassmetastatic cancers of any of the preceding, e.g., prostate, colon,lung or pancreas cancer metastases. The antibody is able to bind toat least a portion of the cancer cells that express Pro115 in themammal and preferably is one that does not induce or that minimizesHAMA response. Preferably, the antibody is effective to destroy orkill Pro115-expressing tumor cells or inhibit the growth of suchtumor cells, in vitro or in vivo, upon binding to Pro115 on thecell. Such an antibody includes a naked anti-Pro115 antibody (notconjugated to any agent). Naked anti-Pro115 antibodies having tumorgrowth inhibition properties in vivo include the antibodiesdescribed in the Experimental Examples below. Naked antibodies thathave cytotoxic or cell growth inhibition properties can be furtherconjugated with a cytotoxic agent to render them even more potentin tumor cell destruction. Cytotoxic properties can be conferred toan anti-Pro115 antibody by, e.g., conjugating the antibody with acytotoxic agent, to form an immunoconjugate as described below. Thecytotoxic agent or a growth inhibitory agent is preferably a smallmolecule. Toxins such as maytansin, maytansinoids, saporin,gelonin, ricin or calicheamicin and analogs or derivatives thereof,are preferable.

[0135] The invention provides a composition comprising ananti-Pro115 antibody of the invention, and a carrier. For thepurposes of treating cancer, compositions can be administered tothe patient in need of such treatment, wherein the composition cancomprise one or more anti-Pro115 antibodies present as animmunoconjugate or as the naked antibody. Further, the compositionscan comprise these antibodies in combination with other therapeuticagents such as cytotoxic or growth inhibitory agents, includingchemotherapeutic agents. The invention also provides formulationscomprising an anti-Pro115 antibody of the invention, and a carrier.The formulation may be a therapeutic formulation comprising apharmaceutically acceptable carrier.

[0136] Another aspect of the invention is isolated nucleic acidsencoding the internalizing anti-Pro115 antibodies. Nucleic acidsencoding both the H and L chains and especially the hypervariableregion residues, chains which encode the native sequence antibodyas well as variants, modifications and humanized versions of theantibody, are encompassed.

[0137] The invention also provides methods useful for treating anPro115-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeuticallyeffective amount of an internalizing anti-Pro115 antibody to themammal. The antibody therapeutic compositions can be administeredshort term (acute) or chronic, or intermittent as directed byphysician. Also provided are methods of inhibiting the growth of,and killing an Pro115 expressing cell. Finally, the invention alsoprovides kits and articles of manufacture comprising at least oneantibody of this invention, preferably at least one internalizinganti-Pro115 antibody of this invention. Kits containing anti-Pro115antibodies find use in detecting Pro115 expression, or intherapeutic or diagnostic assays, e.g., for Pro115 cell killingassays or for purification and/or immunoprecipitation of Pro115from cells. For example, for isolation and purification of Pro115,the kit can contain an anti-Pro115 antibody coupled to a solidsupport, e.g., a tissue culture plate or beads (e.g., sepharosebeads). Kits can be provided which contain antibodies for detectionand quantitation of Pro115 in vitro, e.g. in an ELISA or a Westernblot. Such antibody useful for detection may be provided with alabel such as a fluorescent or radiolabel.

Production of Anti-Pro115 Antibodies

[0138] The following describes exemplary techniques for theproduction of the antibodies useful in the present invention. Someof these techniques are described further in Example 1. The Pro115antigen to be used for production of antibodies may be, e.g., thefull length polypeptide or a portion thereof, including a solubleform of Pro115 lacking the membrane spanning sequence, or syntheticpeptides to selected portions of the protein.

[0139] Alternatively, cells expressing Pro115 at their cell surface(e.g. CHO or NIH-3T3 cells transformed to overexpress Pro115;ovarian, pancreatic, lung, breast or other Pro115-expressing tumorcell line), or membranes prepared from such cells can be used togenerate antibodies. The nucleotide and amino acid sequences ofhuman and murine Pro115 are available as provided above. Pro115 canbe produced recombinantly in and isolated from, prokaryotic cells,e.g., bacterial cells, or eukaryotic cells using standardrecombinant DNA methodology. Pro115 can be expressed as a tagged(e.g., epitope tag) or other fusion protein to facilitate itsisolation as well as its identification in various assays.

[0140] Antibodies or binding proteins that bind to various tags andfusion sequences are available as elaborated below. Other forms ofPro115 useful for generating antibodies will be apparent to thoseskilled in the art.

[0141] Tags

[0142] Various tag polypeptides and their respective antibodies arewell known in the art. Examples include poly-histidine(poly-his) orpoly-histidine-glycine(poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto (Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)); and the Herpes Simplex virusglycoprotein D (gD) tag and its antibody (Paborsky et al., ProteinEngineering, 3(6):547-553 (1990)). The FLAG-peptide (Hopp et al.,BioTechnology, 6:1204-1210 (1988)) is recognized by an anti-FLAG M2monoclonal antibody (Eastman Kodak Co., New Haven, Conn.).Purification of a protein containing the FLAG peptide can beperformed by immunoaffinity chromatography using an affinity matrixcomprising the anti-FLAG M2 monoclonal antibody covalently attachedto agarose (Eastman Kodak Co., New Haven, Conn.). Other tagpolypeptides include the KT3 epitope peptide [Martin et al.,Science, 255:192-194 (1992)1; an .alpha.-tubulin epitope peptide(Skinner et al., J. Biol. Chenz., 266:15163-15166 (1991)); and theT7 gene protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393-6397 (1990)).

[0143] Polyclonal Antibodies

[0144] Polyclonal antibodies are preferably raised in animals,preferably non-human animals, by multiple subcutaneous (sc) orintraperitoneal (ip) injections of the relevant antigen and anadjuvant. It may be useful to conjugate the relevant antigen(especially when synthetic peptides are used) to a protein that isimmunogenic in the species to be immunized. For example, theantigen can be conjugated to keyhole limpet hemocyanin (KLH),serum, bovine thyroglobulin, or soybean trypsin inhibitor, using abifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde,succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where Rand R.sup.1 are different alkyl groups. Conjugates also can be madein recombinant cell culture as protein fusions.

[0145] Animals are immunized against the antigen, immunogenicconjugates, or derivatives by combining, e.g., 5-100 pg of theprotein or conjugate (for rabbits or mice, respectively) with 3volumes of Freund's complete adjuvant and injecting the solutionintradermally at multiple sites. One month later, the animals areboosted with 1/5 to 1/10 the original amount of peptide orconjugate in Freund's complete adjuvant by subcutaneous injectionat multiple sites. Seven to 14 days later, the animals are bled andthe serum is assayed for antibody titer. Animals are boosted untilthe titer plateaus. Also, aggregating agents such as alum aresuitably used to enhance the immune response.

[0146] Monoclonal Antibodies

[0147] Monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), or may bemade by recombinant DNA methods (U.S. Pat. No. 4,816,567). In thehybridoma method, a mouse or other appropriate host animal, such asa hamster, is immunized as described above to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Afterimmunization, lymphocytes are isolated and then fused with a"fusion partner", e.g., a myeloma cell line using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell(Goding, Monoclonal Antibodies. Principles and Practice, pp 103(Academic Press, 1986)).

[0148] The hybridoma cells thus prepared are seeded and grown in asuitable culture medium which medium preferably contains one ormore substances that inhibit the growth or survival of the unfused,fusion partner, e.g., the parental myeloma cells. For example, ifthe parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the selective culturemedium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances preventthe growth of HGPRT-deficient cells.

[0149] Preferred fusion partner myeloma cells are those that fuseefficiently, support stable high-level production of antibody bythe selected antibody-producing cells, and are sensitive to aselective medium that selects against the unfused parental cells.Preferred myeloma cell lines are murine myeloma lines, such asthose derived from MOPC-21 and MPC-11 mouse tumors available fromthe Salk Institute Cell Distribution Center, San Diego, Calif. USA,and SP-2 and derivatives e.g., X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myelomaand mouse-human heteromyeloma cell lines also have been describedfor the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker,Inc., New York, 1987)).

[0150] Culture medium in which hybridoma cells are growing isassayed for production of monoclonal antibodies directed againstthe antigen. Preferably, the binding specificity of monoclonalantibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA).

[0151] The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis described inMunson et al., Anal. Biochem., 107:220 (1980). Once hybridoma cellsthat produce antibodies of the desired specificity, affinity,and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp 103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridomacells may be grown in vivo as ascites tumors in an animal e.g., byi.p. injection of the cells into mice.

[0152] The monoclonal antibodies secreted by the subclones aresuitably separated from the culture medium, ascites fluid, or serumby conventional antibody purification procedures such as, forexample, affinity chromatography (e.g., using protein A or proteinG-Sepharose) or ion-exchange chromatography, hydroxyapatitechromatography, gel electrophoresis, dialysis, etc.

[0153] DNA encoding the monoclonal antibodies is readily isolatedand sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies).The hybridoma cells serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which arethen transformed or transfected into prokaryotic or eukaryotic hostcells such as, e.g., E coli cells, simian COS cells, ChineseHamster Ovary (CHO) cells, or myeloma cells, that do not otherwiseproduce antibody protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibodyinclude Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993)and Phickthun, Immunol. Revs., 130:151-188 (1992).

[0154] Further, the monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using thetechniques described in McCafferty et al., Nature, 348:552-554(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol., 222:581-597 (1991) describe the isolation ofmurine and human antibodies, respectively, using phage libraries.Subsequent publications describe the production of high affinity(nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructingvery large phage libraries (Waterhouse et al., Nuc. Acids. Res.,21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridomatechniques for isolation of monoclonal antibodies.

[0155] The DNA that encodes the antibody may be modified to producechimeric or fusion antibody polypeptides, for example, bysubstituting human heavy chain and light chain constant domain (CHand CL) sequences for the homologous murine sequences (U.S. Pat.No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA,81:6851 (1984)), or by fusing the immunoglobulin coding sequencewith all or part of the coding sequence for a non-immunoglobulinpolypeptide (heterologous polypeptide). The nonimmunoglobulinpolypeptide sequences can substitute for the constant domains of anantibody, or they are substituted for the variable domains of oneantigen-combining site of an antibody to create a chimeric bivalentantibody comprising one antigen-combining site having specificityfor an antigen and another antigen-combining site havingspecificity for a different antigen.

[0156] Humanized Antibodies

[0157] Methods for humanizing non-human antibodies have beendescribed in the art. Preferably, a humanized antibody has one ormore amino acid residues introduced into it from a source which isnonhuman. These non-human amino acid residues are often referred toas "import" residues, which are typically taken from an "import"variable domain. Humanization can be essentially performedfollowing the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized"antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain hasbeen substituted by the corresponding sequence from a non-humanspecies. In practice, humanized antibodies are typically humanantibodies in which some hypervariable region residues and possiblysome FR residues are substituted by residues from analogous sitesin rodent antibodies.

[0158] The choice of human variable domains, both light and heavy,to be used in making the humanized antibodies is very important toreduce antigenicity and HAMA response (human anti-mouse antibody)when the antibody is intended for human therapeutic use. Accordingto the so-called "best-fit" method, the sequence of the variabledomain of a rodent antibody is screened against the entire libraryof known human variable domain sequences. The human V domainsequence which is closest to that of the rodent is identified andthe human framework region (FR) within it accepted for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993);Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method usesa particular framework region derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

[0159] It is further important that antibodies be humanized withretention of high binding affinity for the antigen and otherfavorable biological properties. To achieve this goal, according toa preferred method, humanized antibodies are prepared by a processof analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parentaland humanized sequences. Three-dimensional immunoglobulin modelsare commonly available and are familiar to those skilled in theart.

[0160] Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displayspermits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences sothat the desired antibody characteristic, such as increasedaffinity for the target antigen(s), is achieved. In general, thehypervariable region residues are directly and most substantiallyinvolved in influencing antigen binding.

[0161] Various forms of a humanized anti-Pro115 antibody arecontemplated. For example, the humanized antibody may be anantibody fragment, such as a Fab, which is optionally conjugatedwith one or more cytotoxic agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody may be anintact antibody, such as an intact IgG1 antibody.

[0162] Human Antibodies

[0163] As an alternative to humanization, human antibodies can begenerated. For example, it is now possible to produce transgenicanimals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. For example, it has beendescribed that the homozygous deletion of the antibody heavy-chainjoining region (J.sub.H) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array into suchgerm-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno.,7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all ofGenPharm); 5,545,807; and Alternatively, phage display technology(McCafferty et al., Nature 348:552-553 (1990)) can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domaingenes are cloned in-frame into either a major or minor coat proteingene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of thephage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based onthe functional properties of the antibody also result in selectionof the gene encoding the antibody exhibiting those properties.Thus, the phage mimics some of the properties of the B-cell. Phagedisplay can be performed in a variety of formats, reviewed in,e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571 (1993). Several sources of V-genesegments can be used for phage display. Clackson et al., Nature,352:624-628 (1991) isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genesderived from the spleens of immunized mice. A repertoire of V genesfrom unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can beisolated essentially following the techniques described by Marks etal., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and5,573,905. As discussed above, human antibodies may also begenerated by in vitro activated B cells (see U.S. Pat. Nos.5,567,610 and 5,229,275).

[0164] Antibody Fragments

[0165] In certain circumstances there are advantages of usingantibody fragments, rather than whole antibodies. The smaller sizeof the fragments allows for rapid clearance, and may lead toimproved access to solid tumors. Various techniques have beendeveloped for the production of antibody fragments. Traditionally,these fragments were derived via proteolytic digestion of intactantibodies (see, e.g., Morimoto et al., Journal of Biochemical andBiophysical Methods 24:107-117 (1992); and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produceddirectly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phagelibraries discussed above. Alternatively, Fab'-SH fragments can bedirectly recovered from E. coli and chemically coupled to formF(ab)2 fragments (Carter et al., Bio/Technology 10: 163-167(1992)). According to another approach, F(ab)2 fragments can beisolated directly from recombinant host cell culture. Fab andF(ab)2 fragment with increased in vivo half-life comprising asalvage receptor binding epitope residues are described in U.S.Pat. No. 5,869,046. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. Theantibody of choice may also be a single chain Fv fragment (scFv).See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.5,587,458. Fv and sFv are the only species with intact combiningsites that are devoid of constant regions; thus, they are suitablefor reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector proteinat either the amino or the carboxy terminus of an sFv. See AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment may alsobe a "linear antibody", e.g., as described in U.S. Pat. No.5,641,870 for example. Such linear antibody fragments may bemonospecific or bispecific.

[0166] Bispecific Antibodies

[0167] Bispecific antibodies are antibodies that have bindingspecificities for at least two different epitopes. Exemplarybispecific antibodies may bind to two different epitopes of thePro115 protein. Other such antibodies may combine an Pro115 bindingsite with a binding site for another protein. Alternatively, ananti-Pro115.Arm may be combined with an arm which binds to atriggering molecule on a leukocyte such as a Tcell receptormolecule (e.g. C133), or Fc receptors for IgG (Fc.gamma.R), such asFc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16),so as to focus and localize cellular defense mechanisms to thePro115-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express Pro115. Theseantibodies possess an Pro115-binding arm and an arm which binds thecytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid,ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab)2 bispecific antibodies). WO 96/16673describes a bispecific anti-ErbB2/anti-Fc.gamma.RIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecificanti-ErbB2/anti-Fc.gamma.RI antibody. A bispecificanti-ErbB2/Fc.alpha. antibody is shown in WO98/02463. U.S. Pat. No.5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

[0168] Methods for making bispecific antibodies are known in theart. Traditional production of full length bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-lightchain pairs, where the two chains have different specificities(Millstein et al., Nature, 305:537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. Purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, andthe product yields are low. Similar procedures are disclosed in WO93/08829, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0169] According to a different approach, antibody variable domainswith the desired binding specificities (antibody-antigen combiningsites) are fused to immunoglobulin constant domain sequences.Preferably, the fusion is with an Ig heavy chain constant domain,comprising at least part of the hinge, C.sub.H2, and C.sub.H3regions. It is preferred to have the first heavy-chain constantregion (CHI) containing the site necessary for light chain bonding,present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host cell. Thisprovides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in theconstruction provide the optimum yield of the desired bispecificantibody. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains into a single expressionvector when the expression of at least two polypeptide chains inequal ratios results in high yields or when the ratios have nosignificant affect on the yield of the desired chaincombination.

[0170] Preferably, the bispecific antibodies in this approach arecomposed of a hybrid immunoglobulin heavy chain with a firstbinding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) inthe other arm. It was found that this asymmetric structurefacilitates the separation of the desired bispecific compound fromunwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecificmolecule provides for a facile way of separation. This approach isdisclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

[0171] According to another approach described in U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules canbe engineered to maximize the percentage of heterodimers which arerecovered from recombinant cell culture. The preferred interfacecomprises at least a part of the CH3 domain. In this method, one ormore small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory "cavities" of identical orsimilar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large aminoacid side chains with smaller ones (e.g. alanine or threonine).This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such ashomodimers.

[0172] Bispecific antibodies include cross-linked or"heteroconjugate" antibodies. For example, one of the antibodies inthe heteroconjugate can be coupled to avidin, the other to biotin.Such antibodies have, for example, been proposed to target immunesystem cells to unwanted cells (U.S. Pat. No. 4,676,980), and fortreatment of HIV infection (WO 91/00360, WO 92/200373, and EP03089). Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well knownin the art, and are disclosed in U.S. Pat. No. 4,676,980, alongwith a number of cross-linking techniques.

[0173] Techniques for generating bispecific antibodies fromantibody fragments have also been described in the literature. Forexample, bispecific antibodies can be prepared using chemicallinkage. Brennan et al., Science, 229: 81 (1985) describe aprocedure wherein intact antibodies are proteolytically cleaved togenerate F(ab')2 fragments. These fragments are reduced in thepresence of the dithiol complexing agent, sodium arsenite, tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab' fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab'-TNBderivatives is then reconverted to the Fab'-thiol by reduction withmercaptoethylamine and is mixed with an equimolar amount of theother Fab'-TNB derivative to form the bispecific antibody. Thebispecific antibodies produced can be used as agents for theselective immobilization of enzymes.

[0174] Recent progress has facilitated the direct recovery ofFab'-SH fragments from E. coli, which can be chemically coupled toform bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab')2 molecule. Each Fab' fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing theErbB2 receptor and normal human T cells, as well as trigger thelytic activity of human cytotoxic lymphocytes against human breasttumor targets.

[0175] Various techniques for making and isolating bispecificantibody fragments directly from recombinant cell culture have alsobeen described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.,148(5):1547-1553 (1992). The leucine zipper peptides from the Fosand Jun proteins were linked to the Fab' portions of two differentantibodies by gene fusion. The antibody homodimers were reduced atthe hinge region to form monomers and then re-oxidized to form theantibody heterodimers. This method can also be utilized for theproduction of antibody homodimers.

[0176] The "diabody" technology described by Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a VH connected to a VL by a linker which is tooshort to allow pairing between the two domains on the same chain.Accordingly, the VH and VL domains of one fragment are forced topair with the complementary VL and VH domains of another fragment,thereby forming two antigen-binding sites. Another strategy formaking bispecific antibody fragments by the use of single-chain Fv(sFv) dimers has also been reported. See Gruber et al., J.Immunol., 152:5368 (1994).

[0177] Antibodies with more than two valencies are contemplated.For example, trispecific antibodies can be prepared. Tutt et al. J.Immunol. 147: 60 (1991).

[0178] Multivalent Antibodies

[0179] A multivalent antibody may be internalized (and/orcatabolized) faster than a bivalent antibody by a cell expressingan antigen to which the antibodies bind. The antibodies of thepresent invention can be multivalent antibodies (which are otherthan of the IgM class) with three or more antigen binding sites(e.g. tetravalent antibodies), which can be readily produced byrecombinant expression of nucleic acid encoding the polypeptidechains of the antibody. The multivalent antibody can comprise adimerization domain and three or more antigen binding sites. Thepreferred dimerization domain comprises (or consists of) an Fcregion or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, butpreferably four, antigen binding sites. The multivalent antibodycomprises at least one polypeptide chain (and preferably twopolypeptide chains), wherein the polypeptide chain(s) comprise twoor more variable domains. For instance, the polypeptide chain(s)may comprise VD1(X1n-VD2-(X2)n-Fc, wherein VDI is a first variabledomain, VD2 is a second variable domain, Fc is one polypeptidechain of an Fc region, X1 and X2 represent an amino acid orpolypeptide, and n is 0 or 1. For instance, the polypeptidechain(s) may comprise: VH-CHI-flexible linker-VH-CHI-Fc regionchain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferablyfour) light chain variable domain polypeptides. The multivalentantibody herein may, for instance, comprise from about two to abouteight light chain variable domain polypeptides. The light chainvariable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CLdomain.

[0180] Other Amino Acid Sequence Modifications

[0181] Amino acid sequence modification(s) of the anti-Pro115antibodies described herein are contemplated. For example, it maybe desirable to improve the binding affinity and/or otherbiological properties of the antibody. Amino acid sequence variantsof the anti-Pro115 antibody are prepared by introducing appropriatenucleotide changes into the anti-Pro115 antibody nucleic acid, orby peptide synthesis.

[0182] Such modifications include, for example, deletions from,and/or insertions into, and/or substitutions of, residues withinthe amino acid sequences of the anti-Pro115 antibody. Anycombination of deletion, insertion, and substitution is made toarrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid changes alsomay alter post-translational processes of the anti-Pro115 antibody,such as changing the number or position of glycosylation sites.

[0183] A useful method for identification of certain residues orregions of the anti-Pro115 antibody that are preferred locationsfor mutagenesis is called "alanine scanning mutagenesis" asdescribed by Cunningham and Wells in Science, 244:1081-1085 (1989).Here, a residue or group of target residues within the anti-Pro115antibody are identified (e.g., charged residues such as arg, asp,his, lys, and glu) and replaced by a neutral or negatively chargedamino acid (most preferably alanine or polyalanine) to affect theinteraction of the amino acids with Pro115 antigen.

[0184] Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducingfurther or other variants at, or for, the sites of substitution.Thus, while the site for introducing an amino acid sequencevariation is predetermined, the nature of the mutation per se neednot be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at a target codon or region and the expressed anti-Pro115antibody variants are screened for the desired activity.

[0185] Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an anti-Pro115 antibodywith an N-terminal methionyl residue or the antibody fused to acytotoxic polypeptide. Other insertional variants of theanti-Pro115 antibody molecule include the fusion to the N- orC-terminus of the anti-Pro115 antibody to an enzyme (e.g. forADEPT) or a fusion to a polypeptide which increases the serumhalf-life of the antibody.

[0186] Another type of variant is an amino acid substitutionvariant. These variants have at least one amino acid residue in theanti-Pro115 antibody molecule replaced by a different residue. Thesites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are alsocontemplated. Conservative substitutions are shown in Table I underthe heading of "preferred substitutions". If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated "exemplary substitutions" in the table below,or as further described below in reference to amino acid classes,may be introduced and the products screened for a desiredcharacteristic.

TABLE-US-00003 Amino Acid Substitutions Preferred OriginalExemplary Substitutions Substitutions Ala (A) val; leu; ile Val Arg(R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D)glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp;gln asp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu Leu (L) norleucine; ile; val; met; ala; ileLys (K) arg; gin; asn arg Met (M) leu; phe; ile leu Phe (F) leu;val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) serser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser Phe Val (V)ile; leu; met; phe; ala; leu

[0187] Substantial modifications in the biological properties ofthe antibody are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or(c) the bulk of the side chain. Naturally occurring residues aredivided into groups based on common side-chain properties: [0188](1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutralhydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn,gin, his, lys, arg; (5) residues that influence chain orientation:gly, pro; and (6) aromatic: trp, tyr, phe.

[0189] Non-conservative substitutions will entail exchanging amember of one of these classes for another class. Any cysteineresidue not involved in maintaining the proper conformation of theanti-Pro115 antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule andprevent aberrant crosslinking. Conversely, cysteine bond(s) may beadded to the antibody to improve its stability (particularly wherethe antibody is an antibody fragment such as an Fv fragment).

[0190] A particularly preferred type of substitutional variantinvolves substituting one or more hypervariable region residues ofa parent antibody (e.g. a humanized or human antibody). Generally,the resulting variant(s) selected for further development will haveimproved biological properties relative to the parent antibody fromwhich they are generated. A convenient way for generating suchsubstitutional variants involves affinity maturation using phagedisplay. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino acidsubstitutions at each site. The antibody variants thus generatedare displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged withineach particle. The phage-displayed variants are then screened fortheir biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can beperformed to identify hypervariable region residues contributingsignificantly to antigen binding. Alternatively, or additionally,it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between theantibody and human Pro115. Such contact residues and neighboringresidues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assaysmay be selected for further development.

[0191] Another type of amino acid variant of the antibody altersthe original glycosylation pattern of the antibody. By altering ismeant deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that arenot present in the antibody. Glycosylation of antibodies istypically either N-linked or O-linked. N-linked refers to theattachment of the carbohydrate moiety to the side chain of anasparagine residue. The tripeptide sequences asparagine-X-serineand asparagine-X-threonine, where X is any amino acid exceptproline, are the recognition sequences for enzymatic attachment ofthe carbohydrate moiety to the asparagine side chain. Thus, thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. O-linked glycosylationrefers to the attachment of one of the sugars N-aceylgalactosamine,galactose, or xylose to a hydroxyamino acid, most commonly serineor threonine, although 5-hydroxyproline or 5-hydroxylysine may alsobe used. Addition of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence suchthat it contains one or more of the above-described tripeptidesequences (for N-linked glycosylation sites). The alteration mayalso be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the originalantibody (for O-linked glycosylation sites).

[0192] Nucleic acid molecules encoding amino acid sequence variantsof the anti-Pro115 antibody are prepared by a variety of methodsknown in the art. These methods include, but are not limited to,isolation from a natural source (in the case of naturally occurringamino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier preparednucleic acid molecule encoding a variant or a non-variant versionof the anti-Pro115 antibody.

[0193] It may be desirable to modify the antibody of the inventionwith respect to effector function, e.g. so as to enhanceantigen-dependent cell-mediated cyotoxicity (ADCC) and/orcomplement dependent cytotoxicity (CDC) of the antibody. This maybe achieved by introducing one or more amino acid substitutions inan Fc region of the antibody. Alternatively or additionally,cysteine residue(s) may be introduced in the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediatedcell killing and antibody-dependent cellular cytotoxicity (ADCC).See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies withenhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al.Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby haveenhanced complement lysis and ADCC capabilities. See Stevenson etal. Anti-Cancer Drug Design 3:219-230 (1989).

[0194] To increase the serum half life of the antibody, one mayincorporate a salvage receptor binding epitope into the antibody(especially an antibody fragment) as described in U.S. Pat. No.5,739,277, for example. As used herein, the term "salvage receptorbinding epitope" refers to an epitope of the Fc region of theantibody.

Screening for Antibodies With the Desired Properties

[0195] Techniques for generating antibodies have been describedabove. One may further select antibodies with certain biologicalcharacteristics, as desired.

[0196] The growth inhibitory effects of an anti-Pro115 antibody ofthe invention may be assessed by methods known in the art, e.g.,using cells which express Pro115 either endogenously or followingtransfection with the Pro115 gene. For example, the tumor celllines and Pro115-transfected cells provided in Example 1 below maybe treated with an anti-Pro115 monoclonal antibody of the inventionat various concentrations for a few days (e.g., 2-7) days andstained with crystal violet or MIT or analyzed by some othercolorimetric assay. Another method of measuring proliferation wouldbe by comparing .sup.3H-thymidine uptake by the cells treated inthe presence or absence an anti-Pro115 antibody of the invention.After antibody treatment, the cells are harvested and the amount ofradioactivity incorporated into the DNA quantitated in ascintillation counter. Appropriated positive controls includetreatment of a selected cell line with a growth inhibitory antibodyknown to inhibit growth of that cell line. Growth inhibition oftumor cells in vivo can be determined in various ways such as isdescribed in the Experimental Examples section below. Preferably,the tumor cell is one that over-expresses Pro115. Preferably, theanti-Pro115 antibody will inhibit cell proliferation of anPro115-expressing tumor cell in vitro or in vivo by about 25-100%compared to the untreated tumor cell, more preferably, by about30-100%, and even more preferably by about 50-100% or 70-100%, atan antibody concentration of about 0.5 to 30 .mu.g/ml. Growthinhibition can be measured at an antibody concentration of about0.5 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture, wherethe growth inhibition is determined 1-10 days after exposure of thetumor cells to the antibody. The antibody is growth inhibitory invivo if administration of the anti-Pro115 antibody at about 1.mu.g/kg to about 100 mg/kg body weight results in reduction intumor size or tumor cell proliferation within about 5 days to 3months from the first administration of the antibody, preferablywithin about 5 to 30 days.

[0197] To select for antibodies which induce cell death, loss ofmembrane integrity as indicated by, e.g., propidium iodide (PI),trypan blue or 7AAD uptake may be assessed relative to a control. API uptake assay can be performed in the absence of complement andimmune effector cells. Pro115-expressing tumor cells are incubatedwith medium alone or medium containing of the appropriatemonoclonal antibody at e.g., about 10 .mu.g/ml. The cells areincubated for a 3 day time period. Following each treatment, cellsare washed and aliquoted into 35 mm strainer-capped 12.times.75tubes (1 ml per tube, 3 tubes per treatment group) for removal ofcell clumps. Tubes then receive PI (10 .mu.g/ml). Samples may beanalyzed using a FACSCAN.TM. flow cytometer and FACSCONVERT.TM.CellQuest software (Becton Dickinson). Those antibodies whichinduce statistically significant levels of cell death as determinedby PI uptake may be selected as cell death-inducing antibodies.

[0198] To screen for antibodies which bind to an epitope on Pro115bound by an antibody of interest, e.g., the Pro115 antibodies ofthis invention, a routine cross-blocking assay such as thatdescribe in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed. Thisassay can be used to determine if a test antibody binds the samesite or epitope as an anti-Pro115 antibody of the invention.Alternatively, or additionally, epitope mapping can be performed bymethods known in the art. For example, the antibody sequence can bemutagenized such as by alanine scanning, to identify contactresidues. The mutant antibody is initially tested for binding withpolyclonal antibody to ensure proper folding. In a differentmethod, peptides corresponding to different regions of Pro115 canbe used in competition assays with the test antibodies or with atest antibody and an antibody with a characterized or knownepitope.

[0199] For example, a method to screen for antibodies that bind toan epitope which is bound by an antibody this invention maycomprise combining an Pro115-containing sample with a test antibodyand an antibody of this invention to form a mixture, the level ofPro115 antibody bound to Pro115 in the mixture is then determinedand compared to the level of Pro115 antibody bound in the mixtureto a control mixture, wherein the level of Pro115 antibody bindingto Pro115 in the mixture as compared to the control is indicativeof the test antibody's binding to an epitope that is bound by theanti-Pro115 antibody of this invention. The level of Pro115antibody bound to Pro115 is determined by ELISA. The control may bea positive or negative control or both. For example, the controlmay be a mixture of Pro115, Pro115 antibody of this invention andan antibody known to bind the epitope bound by the Pro115 antibodyof this invention. The anti-Pro115 antibody labeled with a labelsuch as those disclosed herein. The Pro115 may be bound to a solidsupport, e.g., a tissue culture plate or to beads, e.g., sepharosebeads.

Immunoconjugates

[0200] The invention also pertains to therapy with immunoconjugatescomprising an antibody conjugated to an anti-cancer agent such as acytotoxic agent or a growth inhibitory agent.

[0201] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of anantibody and one or more small molecule toxins, such as acalicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

[0202] Maytansine and Maytansinoids

[0203] Preferably, an anti-Pro115 antibody (full length orfragments) of the invention is conjugated to one or moremaytansinoid molecules.

[0204] Maytansinoids are mitototic inhibitors which act byinhibiting tubulin polymerization. Maytansine was first isolatedfrom the cast African shrub Maytenus serrata (U.S. Pat. No.3,896,111). Subsequently, it was discovered that certain microbesalso produce maytansinoids, such as maytansinol and C-3 maytansinolesters (U.S. Pat. No. 4,151,042). Synthetic maytansinol andderivatives and analogues thereof are disclosed, for example, inU.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, thedisclosures of which are hereby expressly incorporated byreference.

[0205] Maytansinoid-Antibody Conjugates

[0206] In an attempt to improve their therapeutic index, maytansineand maytansinoids have been conjugated to antibodies specificallybinding to tumor cell antigens. Immunoconjugates containingmaytansinoids and their therapeutic use are disclosed, for example,in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425235 B1, the disclosures of which are hereby expressly incorporatedby reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623(1996) described immunoconjugates comprising a maytansinoiddesignated DMI linked to the monoclonal antibody C242 directedagainst human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.Cancer Research 52:127-131 (1992) describe immunoconjugates inwhich a maytansinoid was conjugated via a disulfide linker to themurine antibody A7 binding to an antigen on human colon cancer celllines, or to another murine monoclonal antibody TA.1 that binds theHER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3.times.10 5 HER-2 surface antigens percell. The drug conjugate achieved a degree of cytotoxicity similarto the free maytansonid drug, which could be increased byincreasing the number of maytansinoid molecules per antibodymolecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

[0207] Anti-Pro115 Antibody-Maytansinoid Conjugates(Immunoconjugates)

[0208] Anti-Pro115 antibody-maytansinoid conjugates are prepared bychemically linking an anti-Pro115 antibody to a maytansinoidmolecule without significantly diminishing the biological activityof either the antibody or the maytansinoid molecule. An average of3-4 maytansinoid molecules conjugated per antibody molecule hasshown efficacy in enhancing cytotoxicity of target cells withoutnegatively affecting the function or solubility of the antibody,although even one molecule of toxin/antibody would be expected toenhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by knowntechniques or isolated from natural sources. Suitable maytansinoidsare disclosed, for example, in U.S. Pat. No. 5,208,020 and in theother patents and nonpatent publications referred to hereinabove.Preferred maytansinoids are maytansinol and maytansinol analoguesmodified in the aromatic ring or at other positions of themaytansinol molecule, such as various maytansinol esters.

[0209] There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al. Cancer Research 52: 127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Conjugates of the antibody andmaytansinoid may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl(2-pyridyidithio)propionate(SPDP), succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (suchas dimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds(such as his (p-azidobenzoyl)hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).Particularly preferred coupling agents include N-succinimidyl(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J.173:723-737 [1978]) and N-succinimidyl(2-pyridylthio)pentanoate(SPP) to provide for a disulfide linkage.

[0210] The linker may be attached to the maytansinoid molecule atvarious positions, depending on the type of the link. For example,an ester linkage may be formed by reaction with a hydroxyl groupusing conventional coupling techniques. The reaction may occur atthe C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with ahydroxyl group, and the C-20 position having a hydroxyl group.Preferably, the linkage is formed at the C-3 position ofmaytansinol or a maytansinol analogue.

[0211] Calicheamicin

[0212] Another immunoconjugate of interest comprises an anti-Pro115antibody conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S.Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).Structural analogues of calicheamicin which may be used include,but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and.theta..sub.1.sup.I, (Hinman et al. Cancer Research 53: 3336(1993), Lode et al. Cancer Research 5 8: 2925-2928 (1998) and theaforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which isan antifolate. Both calicheamicin and QFA have intracellular sitesof action and do not readily cross the plasma membrane. Therefore,cellular uptake of these agents through antibody mediatedinternalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

[0213] Other antitumor agents that can be conjugated to theanti-Pro115 antibodies of the invention include BCNU,streptozoicin, vincristine and 5-fluorouracil, the family of agentsknown collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.5,877,296). Enzymatically active toxins and fragments thereof whichcan be used include diphtheria A chain, 1 5 nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, saponaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycinand the tricothecenes. See, for example, WO 93/21232 published Oct.28, 1993. The present invention further contemplates animmunoconjugate formed between an antibody and a compound withnucleolytic activity (e.g. a ribonuclease or a DNA endonucleasesuch as a deoxyribonuclease; DNase).

[0214] For selective destruction of the tumor, the antibody maycomprise a highly radioactive atom. A variety of radioactiveisotopes are available for the production of radioconjugatedanti-Prof115 antibodies. Examples include At.sup.211, I.sup.131,I.sup.125, In.sup.111, Y.sup.90, Re.sup.186, Re.sup.188,Sm.sup.153, Bi.sup.212, P.sup.32, and radioactive isotopes of Lu.When the conjugate is used for diagnosis, it may comprise aradioactive atom for scintigraphic studies, for example Tc.sup.99Mor I.sup.123, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0215] The radio- or other labels may be incorporated in theconjugate in known ways. For example, the peptide may bebiosynthesized or may be synthesized by chemical amino acidsynthesis using suitable amino acid precursors involving, forexample, fluorine-19 in place of hydrogen. Labels such asTc.sup.99M, I.sup.123, In.sup.111, Re.sup.186, Re.sup.188, can beattached via a cysteine residue in the peptide. Yttrium-90 can beattached via a lysine residue. The IODOGEN method (Fraker et al(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used toincorporate iodine "Monoclonal Antibodies in Immunoscintigraphy"(Chatal, CRC Press 1989) describes other methods in detail.

[0216] Conjugates of the antibody and cytotoxic agent may be madeusing a variety of bifunctional protein coupling agents such asN-succinimidyl(2-pyridyldithio)propionate (SPDP),succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (suchas dimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds(such as bis (p-azidobenzoyl)hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described inVitetta et al. Science 238: 1098 (1987). Carbon labeled1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO 94/11026. The linker may bea "cleavable linker" facilitating release of the cytotoxic drug inthe cell. For example, an acid-labile linker, peptidase-sensitivelinker, photolabile linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992); U.S. Pat.No. 5,208,020) may be used.

[0217] Alternatively, a fusion protein comprising the anti-Pro115antibody and cytotoxic agent may be made, e.g. by recombinanttechniques or peptide synthesis. The length of DNA may compriserespective regions encoding the two portions of the conjugateeither adjacent one another or separated by a region encoding alinker peptide which does not destroy the desired properties of theconjugate.

[0218] In addition, the antibody may be conjugated to a "receptor"(such streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation usinga clearing agent and then administration of a "ligand" (e.g.avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide).

Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

[0219] The antibodies of the present invention may also be used inADEPT by conjugating the antibody to a prodrug-activating enzymewhich converts a prodrug (e.g. a peptidyl chemotherapeutic agent,see W081/01145) to an active anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No. 4,975,278.

[0220] The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way soas to covert it into its more active, cytotoxic form. Enzymes thatare useful in the method of this invention include, but are notlimited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase usefulfor converting sulfate-containing prodrugs into free drugs;cytosine deaminase useful for converting non-toxic fluorocytosineinto the anti-cancer drug, 5-fluorouracil; proteases, such asserratia protease, thermolysin, subtilisin, carboxypeptidases andcathepsins (such as cathepsins B and L), that are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymessuch as O-galactosidase and neuraminidase useful for convertingglycosylated prodrugs into free drugs; P-lactamase useful forconverting drugs derivatized with P-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. Alternatively, antibodies with enzymatic activity,also known in the art as "abzymes", can be used to convert theprodrugs of the invention into free active drugs (see, e.g.,Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates canbe prepared as described herein for delivery of the abzyme to atumor cell population. The enzymes of this invention can becovalently bound to the anti-Pro115 antibodies by techniques wellknown in the art such as the use of the heterobifunctionalcrosslinking reagents discussed above.

[0221] Alternatively, fusion proteins comprising at least theantigen binding region of an antibody of the invention linked to atleast a functionally active portion of an enzyme of the inventioncan be constructed using recombinant DNA techniques well known inthe art (see, e.g., Neuberger et al., Nature, 312: 604-608(1984).

Other Antibody Modifications

[0222] Other modifications of the antibody are contemplated herein.For example, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques orby interfacial polymerization (for example, hydroxymethylcelluloseor gelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosedin Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.,Ed., (1980).

[0223] The anti-Pro115 antibodies disclosed herein may also beformulated as immunoliposomes. A "liposome" is a small vesiclecomposed of various types of lipids, phospholipids and/orsurfactant which is useful for delivery of a drug to a mammal. Thecomponents of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biologicalmembranes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al.,Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and W097/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab' fragments of the antibody of the present inventioncan be conjugated to the liposomes as described in Martin et al. J.Biol. Chem. 257: 286-288 (1982) via a disulfide interchangereaction. A chemotherapeutic agent is optionally contained withinthe liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484 (1989).

Vectors, Host Cells, and Recombinant Methods

[0224] The invention also provides isolated nucleic acid moleculeencoding the humanized anti-Pro115 antibody, vectors and host cellscomprising the nucleic acid, and recombinant techniques for theproduction of the antibody. For recombinant production of theantibody, the nucleic acid molecule encoding it is isolated andinserted into a replicable vector for further cloning(amplification of the DNA) or inserted into a vector in operablelinkage with a promoter for expression. DNA encoding the monoclonalantibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capableof binding specifically to nucleic acid molecules encoding theheavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancerelement, a promoter, and a transcription termination sequence.

[0225] Signal Sequence Component

[0226] The anti-Pro115 antibody of this invention may be producedrecombinantly not only directly, but also as a fusion polypeptidewith a heterologous polypeptide, which is preferably a signalsequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) bythe host cell. For prokaryotic host cells that do not recognize andprocess the native anti-Pro115 antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected,for example, from the group of the alkaline phosphatase,penicillinase, 1pp, or heat-stable enterotoxin II leaders. Foryeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, oc factor leader (includingSaccharomyces and Kluyveromyces cc-factor leaders), or acidphosphatase leader, the C albicans glucoamylase leader, or thesignal described in WO 90/13646. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available. The DNA forsuch precursor region is ligated in reading frame to DNA encodingthe anti-Pro115 antibody.

[0227] Origin of Replication

[0228] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or moreselected host cells. Generally, in cloning vectors this sequence isone that enables the vector to replicate independently of the hostchromosomal DNA, and includes origins of replication orautonomously replicating sequences. Such sequences are well knownfor a variety of bacteria, yeast, and viruses. The origin ofreplication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2.mu. plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally,the origin of replication component is not needed for mammalianexpression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter).

[0229] Selection Gene Component

[0230] Expression and cloning vectors may contain a selection gene,also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. One example of a selection schemeutilizes a drug to arrest growth of a host cell. Those cells thatare successfully transformed with a heterologous gene produce aprotein conferring drug resistance and thus survive the selectionregimen. Examples of such dominant selection use the drugsneomycin, mycophenolic acid and hygromycin.

[0231] Another example of suitable selectable markers for mammaliancells are those that enable the identification of cells competentto take up the anti-Pro115 antibody nucleic acid, such as DHFR,thymidine kinase, metallothionein-I and -11, preferably primatemetallothionein genes, adenosine deaminase, ornithinedecarboxylase, etc. For example, cells transformed with the DHFRselection gene are first identified by culturing all of thetransformants in a culture medium that contains methotrexate (Mtx),a competitive antagonist of DHFR. An appropriate host cell whenwild-type DHFR is employed is the Chinese hamster ovary (CHO) cellline deficient in DHFR activity (e.g., ATCC CRL-9096).

[0232] Alternatively, host cells (particularly wild-type hosts thatcontain endogenous DHFR) transformed or co-transformed with DNAsequences encoding anti-Pro115 antibody, wild-type DHFR protein,and another selectable marker such as aminoglycoside3'-phosphotransferase (APH) can be selected by cell growth inmedium containing a selection agent for the selectable marker suchas an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, orG418. See U.S. Pat. No. 4,965,199.

[0233] A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid YRp7 (Stinchcomb et al., Nature,282:39 (1979)). The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan,for example, ATCC No. 44076 or PEP4 Jones, Genetics, 85:12 (1977).The presence of the trp1 lesion in the yeast host cell genome thenprovides an effective environment for detecting transformation bygrowth in the absence of tryptophan. Similarly, Leu2-deficientyeast strains (ATCC 20,622 or 38,626) are complemented by knownplasmids bearing the Leu2 gene.

[0234] In addition, vectors derived from the 1.6 pm circularplasmid pKDI can be used for transformation of Kluyveromycesyeasts. Alternatively, an expression system for large-scaleproduction of recombinant calf chymosin was reported for K. lactis.Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also beendisclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).

[0235] Promoter Component

[0236] Expression and cloning vectors usually contain a promoterthat is recognized by the host organism and is operably linked tothe anti-Pro115 antibody nucleic acid. Promoters suitable for usewith prokaryotic hosts include the phoA promoter , P-lactamase andlactose promoter systems, alkaline phosphatase promoter, atryptophan (trp) promoter system, and hybrid promoters such as thetac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will containa Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the anti-Pro115 antibody.

[0237] Promoter sequences are known for eukaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to30 bases upstream from the site where transcription is initiated.Another sequence found 70 to 80 bases upstream from the start oftranscription of many genes is a CNCAAT region where N may be anynucleotide. At the 3' end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail tothe 3' end of the coding sequence. All of these sequences aresuitably inserted into eukaryotic expression vectors. Examples ofsuitable promoter sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase, glucosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0238] Other yeast promoters, which are inducible promoters havingthe additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associatedwith nitrogen metabolism, metallothionein, glyceraldehyde phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization. Suitable vectors and promoters for use in yeastexpression are further described in EP 73,657. Yeast enhancers alsoare advantageously used with yeast promoters.

[0239] Anti-Pro115 antibody transcription from vectors in mammalianhost cells is controlled, for example, by promoters obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, aviansarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus andmost preferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter,from heat-shock promoters, provided such promoters are compatiblewith the host cell systems.

[0240] The early and late promoters of the SV40 virus areconveniently obtained as an SV40 restriction fragment that alsocontains the SV40 viral origin of replication. The immediate earlypromoter of the human cytomegalovirus is conveniently obtained as aHindIll E restriction fragment. A system for expressing DNA inmammalian hosts using the bovine papilloma virus as a vector isdisclosed in U.S. Pat. No. 4,419,446. A modification of this systemis described in U.S. Pat. No. 4,601,978. See also Reyes et al.,Nature 297:598-601 (1982) on expression of human P-interferon cDNAin mouse cells under the control of a thymidine kinase promoterfrom herpes simplex virus. Alternatively, the Rous Sarcoma Viruslong terminal repeat can be used as the promoter.

[0241] Enhancer Element Component

[0242] Transcription of a DNA encoding the anti-Pro115 antibody ofthis invention by higher eukaryotes is often increased by insertingan enhancer sequence into the vector. Many enhancer sequences arenow known from mammalian genes (globin, elastase, albumin,.alpha.-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhanceron the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer maybe spliced into the vector at a position 5' or 3' to theanti-Pro115 antibody-encoding sequence, but is preferably locatedat a site 5' from the promoter.

[0243] Transcription Termination Component

[0244] Expression vectors used in eukaryotic host cells (yeast,fungi, insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary forthe termination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5' and, occasionally 3'untranslated regions of eukaryotic or viral DNAs or cDNAs. Theseregions contain nucleotide segments transcribed as polyadenylatedfragments in the untranslated portion of the mRNA encodinganti-Pro115 antibody. One useful transcription terminationcomponent is the bovine growth hormone polyadenylation region. SeeWO 94/11026 and the expression vector disclosed therein.

[0245] Selection and Transformation of Host Cells

[0246] Suitable host cells for cloning or expressing the DNA in thevectors herein are the prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,Salmonella typhimurium, Serratia, e.g., Serratia marcescans, andShigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. One preferred E. coli cloning host is E. coli 294(ATCC 31,446), although other strains such as E. coli B, E. coliX1776 (ATCC 31,537), and E. coli W31 10 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

[0247] Full length antibody, antibody fragments, and antibodyfusion proteins can be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed, such as whenthe therapeutic antibody is conjugated to a cytotoxic agent (e.g.,a toxin) and the immunoconjugate by itself shows effectiveness intumor cell destruction. Full length antibodies have greater halflife in circulation. Production in E. coli is faster and more costefficient. For expression of antibody fragments and polypeptides inbacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523(Simmons et al.) which describes translation initiation region(TIR) and signal sequences for optimizing expression and secretion,these patents incorporated herein by reference. After expression,the antibody is isolated from the E. coli cell paste in a solublefraction and can be purified through, e.g., a protein A or G columndepending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., inCHO cells.

[0248] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hostsfor anti-Pro115 antibody-encoding vectors. Saccharomycescerevisiae, or common baker's yeast, is the most commonly usedamong lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe; Kluyveromyceshosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces such as Schwanniomyces occidentalis; andfilamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A.niger.

[0249] Suitable host cells for the expression of glycosylatedanti-Pro115 antibody are derived from multicellular organisms.Examples of invertebrate cells include plant and insect cells.Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruit fly), andBombyx mori have been identified. A variety of viral strains fortransfection are publicly available, e.g., the L-1 variant ofAutographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

[0250] Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, Arabidopsis and tobacco can also be utilized ashosts. Cloning and expression vectors useful in the production ofproteins in plant cell culture are known to those of skill in theart. See e.g. Hiatt et al., Nature (1989) 342: 76-78, Owen et al.(1992) Bio/Technology 10: 790-794, Artsaenko et al. (1995) ThePlant J 8: 745-750, and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0251] However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) hasbecome a routine procedure. Examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)) ; baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)) ; mouse sertoli cells (TM4, Mather,Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL1587);human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidneycells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells(Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1);TRI cells (Mather et al., Annals N. Y Acad. Sci. 383:44-68 (1982));MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0252] Host cells are transformed with the above-describedexpression or cloning vectors for anti-Pro115 antibody productionand cultured in conventional nutrient media modified as appropriatefor inducing promoters, selecting transformants, or amplifying thegenes encoding the desired sequences.

[0253] Culturing Host Cells

[0254] The host cells used to produce the anti-Pro115 antibody ofthis invention may be cultured in a variety of media. Commerciallyavailable media such as Ham's FIO (Sigma), Minimal Essential Medium(MEM)(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium (DMEM)(Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz.58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used asculture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts(such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN.TM. drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or anequivalent energy source. Any other necessary supplements may alsobe included at appropriate concentrations that would be known tothose skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

[0255] Purification of Anti-Pro115 Antibody

[0256] When using recombinant techniques, the antibody can beproduced intracellularly, in the periplasmic space, or directlysecreted into the medium. If the antibody is producedintracellularly, as a first step, the particulate debris, eitherhost cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Carter et al., Bio/Technology10: 163-167 (1992) describe a procedure for isolating antibodieswhich are secreted to the periplasmic space of E coli. Briefly,cell paste is thawed in the presence of sodium acetate (pH 3.5),EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systemsare generally first concentrated using a commercially availableprotein concentration filter, for example, an Amicon or MilliporePellicon ultrafiltration unit. A protease inhibitor such as PMSFmay be included in any of the foregoing steps to inhibitproteolysis and antibiotics may be included to prevent the growthof adventitious contaminants.

[0257] The antibody composition prepared from the cells can bepurified using, for example, hydroxyapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography, withaffinity chromatography being the preferred purification technique.The suitability of protein A as an affinity ligand depends on thespecies and isotype of any immunoglobulin Fc domain that is presentin the antibody. Protein A can be used to purify antibodies thatare based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human .gamma.3 (Guss etal., EMBO J. 5:15671575 (1986)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled poreglass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose.Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation onan ion-exchange column, ethanol precipitation, Reverse Phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE.TM.chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SIDS-PAGE, andammonium sulfate precipitation are also available depending on theantibody to be recovered.

[0258] Following any preliminary purification step(s), the mixturecomprising the antibody of interest and contaminants may besubjected to low pH hydrophobic interaction chromatography using anelution buffer at a pH between about 2.5-4.5, preferably performedat low salt concentrations (e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

[0259] Pharmaceutical formulations of the antibodies used inaccordance with the present invention are prepared for storage bymixing an antibody having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980)), in the form of lyophilized formulations oraqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as acetate, Tris, phosphate,citrate, and other organic acids; antioxidants including ascorbicacid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride,benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propyl paraben; catechol; resorcinol;cyclohexanol; 3-pentanol, and mcresol); low molecular weight (lessthan about 10 residues) polypeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrollidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,or dextrins; chelating agents such as EDTA; tonicifiers such astrehalose and sodium chloride; sugars such as sucrose, mannitol,trehalose or sorbitol; surfactant such as polysorbate; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN.TM.,PLURONICS.TM. or polyethylene glycol (PEG). The antibody preferablycomprises the antibody at a concentration of between 5-200 mg/ml,preferably between 10-100 mg/ml.

[0260] The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do notadversely affect each other. For example, in addition to theanti-Pro115 antibody which internalizes, it may be desirable toinclude in the one formulation, an additional antibody, e.g. asecond anti-Pro115 antibody which binds a different epitope onPro115, or an antibody to some other target such as a growth factorthat affects the growth of the particular cancer. Alternatively, oradditionally, the composition may further comprise achemotherapeutic agent, cytotoxic agent, cytokine, growthinhibitory agent, anti-hormonal agent, and/or cardioprotectant.Such molecules are suitably present in combination in amounts thatare effective for the purpose intended.

[0261] The active ingredients may also be entrapped inmicrocapsules prepared, for example, by coacervation techniques orby interfacial polymerization, for example, hydroxymethylcelluloseor gelatin microcapsules and poly-(methylmethacylate)microcapsules, respectively, in colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Suchtechniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

[0262] Sustained-release preparations may be prepared. Suitableexamples of sustained-release preparations include semi-permeablematrices of solid hydrophobic polymers containing the antibody,which matrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamicacid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinylacetate, degradable lactic acid-glycolic acid copolymers such asthe LUPRON DEPOT.TM. (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(-) hydroxybutyric acid.

[0263] The formulations to be used for in vivo administration mustbe sterile. This is readily accomplished by filtration throughsterile filtration membranes.

Methods and Treatment Using Anti-Pro115 Antibodies

[0264] According to the present invention, the anti-Pro115 antibodythat internalizes upon binding Pro115 on a cell surface is used totreat a subject in need thereof having a cancer characterized byPro115-expressing cancer cells, in particular, prostate, colon,lung or pancreas cancer, and associated metastases.

[0265] The cancer will generally comprise Pro115-expressing cells,such that the anti-Pro115 antibody is able to bind thereto. Whilethe cancer may be characterized by overexpression of the Pro115molecule, the present application further provides a method fortreating cancer which is not considered to be anPro115-overexpressing cancer.

[0266] This invention also relates to methods for detecting cellsor tissues which overexpress Pro115 and to diagnostic kits usefulin detecting cells or tissues expressing Pro115 or in detectingPro115 in bodily fluids from a patient. Bodily fluids includeblood, serum, plasma, urine, ascites, peritoneal wash, saliva,sputum, seminal fluids, mucous membrane secretions, and otherbodily excretions such as stool. The methods may comprise combininga cell-containing test sample with an antibody of this invention,assaying the test sample for antibody binding to cells in the testsample and comparing the level of antibody binding in the testsample to the level of antibody binding in a control sample ofcells. A suitable control is, e.g., a sample of normal cells of thesame type as the test sample or a cell sample known to be free ofPro115 overexpressing cells. A level of Pro115 binding higher thanthat of such a control sample would be indicative of the testsample containing cells that overexpress Pro115. Alternatively thecontrol may be a sample of cells known to contain cells thatoverexpress Pro115. In such a case, a level of Pro115 antibodybinding in the test sample that is similar to, or in excess of,that of the control sample would be indicative of the test samplecontaining cells that overexpress Pro115.

[0267] Pro115 overexpression may be detected with a variousdiagnostic assays. For example, over expression of Pro115 may beassayed by immunohistochemistry (IHC). Parrafin embedded tissuesections from a tumor biopsy may be subjected to the IHC assay andaccorded an Pro115 protein staining intensity criteria asfollows.

[0268] Score 0 no staining is observed or membrane staining isobserved in less than 10% of tumor cells.

[0269] Score 1+ a faint/barely perceptible membrane staining isdetected in more than 10% of the tumor cells. The cells are onlystained in part of their membrane.

[0270] Score 2+ a weak to moderate complete membrane staining isobserved in more than 10% of the tumor cells.

[0271] Score 3+ a moderate to strong complete membrane staining isobserved in more than 10% of the tumor cells.

[0272] Those tumors with 0 or 1+ scores for Pro115 expression maybe characterized as not overexpressing Pro115, whereas those tumorswith 2+ or 3+ scores may be characterized as overexpressingPro115.

[0273] Alternatively, or additionally, FISH assays such as theINFORM.TM. (sold by Ventana, Arizona) or PATHVISION.TM. (VySiS,Illinois) may be carried out on formalin-fixed, paraffin-embeddedtumor tissue to determine the extent (if any) of Pro115overexpression in the tumor. Pro115 overexpression or amplificationmay be evaluated using an in vivo diagnostic assay, e.g. byadministering a molecule (such as an antibody of this invention)which binds Pro115 and which is labeled with a detectable label(e.g. a radioactive isotope or a fluorescent label) and externallyscanning the patient for localization of the label.

[0274] A sample suspected of containing cells expressing oroverexpressing Pro115 is combined with the antibodies of thisinvention under conditions suitable for the specific binding of theantibodies to Pro115. Binding and/or internalizing the Pro115antibodies of this invention is indicative of the cells expressingPro115. The level of binding may be determined and compared to asuitable control, wherein an elevated level of bound Pro115 ascompared to the control is indicative of Pro115 overexpression. Thesample suspected of containing cells overexpressing Pro115 may be acancer cell sample, particularly a sample of prostate, colon, lungor pancreas cancer. A serum sample from a subject may also beassayed for levels of Pro115 by combining a serum sample from asubject with an Pro115 antibody of this invention, determining thelevel of Pro115 bound to the antibody and comparing the level to acontrol, wherein an elevated level of Pro115 in the serum of thepatient as compared to a control is indicative of overexpression ofPro115 by cells in the patient. The subject may have a cancer suchas prostate, colon, lung or pancreas cancer.

[0275] Currently, depending on the stage of the cancer, prostate,colon, lung or pancreas cancer treatment involves one or acombination of the following therapies: surgery to remove thecancerous tissue, radiation therapy, androgen deprivation (e.g.,hormonal therapy), and chemotherapy. Anti-Pro115 antibody therapymay be especially desirable in elderly patients who do not toleratethe toxicity and side effects of chemotherapy well, in metastaticdisease where radiation therapy has limited usefulness, and for themanagement of prostatic carcinoma that is resistant to androgendeprivation treatment. The tumor targeting and internalizinganti-Pro115 antibodies of the invention are useful to alleviatePro115-expressing cancers, e.g., prostate, colon, lung or pancreascancers upon initial diagnosis of the disease or during relapse.For therapeutic applications, the anti-Pro115 antibody can be usedalone, or in combination therapy with, e.g., hormones,antiangiogens, or radiolabelled compounds, or with surgery,cryotherapy, and/or radiotherapy, notably for prostate, colon, lungor pancreas cancers, also particularly where shed cells cannot bereached. Anti-Pro115 antibody treatment can be administered inconjunction with other forms of conventional therapy, eitherconsecutively with, pre- or post-conventional therapy,Chemotherapeutic drugs such as Taxotere.RTM. (docetaxel),Taxol.RTM. (paclitaxel), estramustine and mitoxantrone are used intreating metastatic and hormone refractory prostate, colon, lung orpancreas cancer, in particular, in good risk patients. In thepresent method of the invention for treating or alleviating cancer,in particular, androgen independent and/or metastatic prostate,colon, lung or pancreas cancer, the cancer patient can beadministered anti-Pro115 antibody in conjunction with treatmentwith the one or more of the preceding chemotherapeutic agents. Inparticular, combination therapy with palictaxel and modifiedderivatives (see, e.g., EP0600517) is contemplated. The anti-Pro115antibody will be administered with a therapeutically effective doseof the chemotherapeutic agent. The anti-Pro115 antibody may also beadministered in conjunction with chemotherapy to enhance theactivity and efficacy of the chemotherapeutic agent, e.g.,paclitaxel. The Physicians' Desk Reference (PDR) discloses dosagesof these agents that have been used in treatment of variouscancers. The dosing regimen and dosages of these aforementionedchemotherapeutic drugs that are therapeutically effective willdepend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in theart and can be determined by the physician.

[0276] Particularly, an immunoconjugate comprising the anti-Pro115antibody conjugated with a cytotoxic agent may be administered tothe patient. Preferably, the immunoconjugate bound to the Pro115protein is internalized by the cell, resulting in increasedtherapeutic efficacy of the immunoconjugate in killing the cancercell to which it binds. Preferably, the cytotoxic agent targets orinterferes with the nucleic acid in the cancer cell. Examples ofsuch cytotoxic agents are described above and include maytansin,maytansinoids, saporin, gelonin, ricin, calicheamicin,ribonucleases and DNA endonucleases.

[0277] The anti-Pro115 antibodies or immunoconjugates areadministered to a human patient, in accord with known methods, suchas intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The antibodies orimmunoconjugates may be injected directly into the tumor mass.Intravenous or subcutaneous administration of the antibody ispreferred. Other therapeutic regimens may be combined with theadministration of the anti-Pro115 antibody.

[0278] The combined administration includes co-administration,using separate formulations or a single pharmaceutical formulation,and consecutive administration in either order, wherein preferablythere is a time period while both (or all) active agentssimultaneously exert their biological activities. Preferably suchcombined therapy results in a synergistic therapeutic effect.

[0279] It may also be desirable to combine administration of theanti-Pro115 antibody or antibodies, with administration of anantibody directed against another tumor antigen associated with theparticular cancer. As such, this invention is also directed to anantibody "cocktail" comprising one or more antibodies of thisinvention and at least one other antibody which binds another tumorantigen associated with the Pro115-expressing tumor cells. Thecocktail may also comprise antibodies that are directed to otherepitopes of Pro115. Preferably the other antibodies do notinterfere with the binding and or internalization of the antibodiesof this invention.

[0280] The antibody therapeutic treatment method of the presentinvention may involve the combined administration of an anti-Pro115antibody (or antibodies) and one or more chemotherapeutic agents orgrowth inhibitory agents, including co-administration of cocktailsof different chemotherapeutic agents. Chemotherapeutic agentsinclude, e.g., estramustine phosphate, prednimustine, cisplatin,5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea andhydroxyureataxanes (such as paclitaxel and doxetaxel) and/oranthracycline antibiotics. Preparation and dosing schedules forsuch chemotherapeutic agents may be used according tomanufacturers' instructions or as determined empirically by theskilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992).

[0281] The antibody may be combined with an anti-hormonal compound;e.g., an anti-estrogen compound such as tamoxifen; ananti-progesterone such as onapristone (see, EP 616 812); or ananti-androgen such as flutamide, in dosages known for suchmolecules. Where the cancer to be treated is androgen independentcancer, the patient may previously have been subjected toanti-androgen therapy and, after the cancer becomes androgenindependent, the anti-Pro115 antibody (and optionally other agentsas described herein) may be administered to the patient.

[0282] Sometimes, it may be beneficial to also co-administer acardioprotectant (to prevent or reduce myocardial dysfunctionassociated with the therapy) or one or more cytokines to thepatient. In addition to the above therapeutic regimes, the patientmay be subjected to surgical removal of cancer cells and/orradiation therapy, before, simultaneously with, or post antibodytherapy. Suitable dosages for any of the above co-administeredagents are those presently used and may be lowered due to thecombined action (synergy) of the agent and anti-Pro115antibody.

[0283] For the prevention or treatment of disease, the dosage andmode of administration will be chosen by the physician according toknown criteria. The appropriate dosage of antibody will depend onthe type of disease to be treated, as defined above, the severityand course of the disease, whether the antibody is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretionof the attending physician. The antibody is suitably administeredto the patient at one time or over a series of treatments.Preferably, the antibody is administered by intravenous infusion orby subcutaneous injections. Depending on the type and severity ofthe disease, about 1 pg/kg to about 50 mg/kg body weight (e.g.about 0.1-15 mg/kg/dose) of antibody can be an initial candidatedosage for administration to the patient, whether, for example, byone or more separate administrations, or by continuous infusion. Adosing regimen can comprise administering an initial loading doseof about 4 mg/kg, followed by a weekly maintenance dose of about 2mg/kg of the anti-Pro115 antibody. However, other dosage regimensmay be useful. A typical daily dosage might range from about 1pg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until adesired suppression of disease symptoms occurs. The progress ofthis therapy can be readily monitored by conventional methods andassays and based on criteria known to the physician or otherpersons of skill in the art.

[0284] Aside from administration of the antibody protein to thepatient, the present application contemplates administration of theantibody by gene therapy. Such administration of a nucleic acidmolecule encoding the antibody is encompassed by the expression"administering a therapeutically effective amount of an antibody".See, for example, WO 96/07321 published Mar. 14, 1996 concerningthe use of gene therapy to generate intracellular antibodies.

[0285] There are two major approaches to introducing the nucleicacid molecule (optionally contained in a vector) into the patient'scells; in vivo and ex vivo. For in vivo delivery the nucleic acidmolecule is injected directly into the patient, usually at the sitewhere the antibody is required. For ex vivo treatment, thepatient's cells are removed, the nucleic acid molecule isintroduced into these isolated cells and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). Thereare a variety of techniques available for introducing nucleic acidmolecules into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells invitro, or in vivo in the cells of the intended host. Techniquessuitable for the transfer of nucleic acid into mammalian cells invitro include the use of liposomes, electroporation,microinjection, cell fusion, DEAE-dextran, the calcium phosphateprecipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retroviral vector.

[0286] The currently preferred in vivo nucleic acid moleculetransfer techniques include transfection with viral vectors (suchas adenovirus, Herpes simplex I virus, or adeno-associated virus)and lipid-based systems (useful lipids for lipid-mediated transferof the gene are DOTMA, DOPE and DC-Chol, for example). For reviewof the currently known gene marking and gene therapy protocols seeAnderson et at., Science 256:808-813 (1992). See also WO 93/25673and the references cited therein.

Articles of Manufacture and Kits

[0287] The invention also relates to an article of manufacturecontaining materials useful for the detection for Pro115overexpressing cells and/or the treatment of Pro115 expressingcancer, in particular prostate, colon, lung or pancreas cancer. Thearticle of manufacture comprises a container and a compositioncontained therein comprising an antibody of this invention. Thecomposition may further comprise a carrier. The article ofmanufacture may also comprise a label or package insert on orassociated with the container. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for detectingPro115 expressing cells and/or treating a cancer condition and mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an anti-Pro115 antibody of the invention. The labelor package insert indicates that the composition is used fordetecting Pro115 expressing cells and/or for treating prostate,colon, lung or pancreas cancer, in a patient in need thereof. Thelabel or package insert may further comprise instructions foradministering the antibody composition to a cancer patient.Additionally, the article of manufacture may further comprise asecond container comprising a substance which detects the antibodyof this invention, e.g., a second antibody which binds to theantibodies of this invention. The substance may be labeled with adetectable label such as those disclosed herein. The secondcontainer may contain e.g., a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution.The article of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

[0288] Kits are also provided that are useful for various purposes,e.g., for Pro115 cell killing assays, for purification orimmunoprecipitation of Pro115 from cells or for detecting thepresence of Pro115 in a serum sample or detecting the presence ofPro115-expressing cells in a cell sample. For isolation andpurification of Pro115, the kit can contain an anti-Pro115 antibodycoupled to a solid support, e.g., a tissue culture plate or beads(e.g., sepharose beads). Kits can be provided which contain theantibodies for detection and quantitation of Pro115 in vitro, e.g.in an ELISA or a Western blot. As with the article of manufacture,the kit comprises a container and a composition contained thereincomprising an antibody of this invention. The kit may furthercomprise a label or package insert on or associated with thecontainer. The kits may comprise additional components, e.g.,diluents and buffers, substances which bind to the antibodies ofthis invention, e.g., a second antibody which may comprise a labelsuch as those disclosed herein, e.g., a radiolabel, fluorescentlabel, or enzyme, or the kit may also comprise control antibodies.The additional components may be within separate containers withinthe kit. The label or package insert may provide a description ofthe composition as well as instructions for the intended in vitroor diagnostic use.

Examples

Example 1

Production and Isolation of Monoclonal Antibody ProducingHybridomas

[0289] The following MAb/hybridomas of the present invention aredescribed below: Pro115.A1, Pro115.A2, Pro115.A3, Pro115.A4,Pro115.A5, Pro115.A6, Pro115.A7, Pro115.A8, Pro115.A9, Pro115.A10,Pro115.A11, Pro115.A12, Pro115.A13, Pro115.A14, Pro115.A15,Pro115.A16, Pro115.A17, Pro115.A18, Pro115.A19, Pro115.A20,Pro115.A21, Pro115.A22, Pro115.A23, Pro115.A24, Pro115.A25,Pro115.A101.1, Pro115.A102.1, Pro115.A103.1, Pro115.A104.1,Pro115.A106.1, Pro115.A107.1, Pro115.A108.1, Pro115.B1, Pro115.B2,Pro115.B3, Pro115.B4, Pro115.B5, Pro115.B6, Pro115.B7, Pro115.B8,Pro115.B9, Pro115.B10, Pro115.B11, Pro115.B12, Pro115.B13,Pro115.B14, Pro115.B15, Pro115.B16, Pro115.B17, Pro115.B18,Pro115.B19, Pro115.B20, Pro115.B21, Pro115.B22, Pro115.B23,Pro115.B24, Pro115.B25, Pro115.B26, Pro115.B27, Pro115.B28,Pro115.B29, Pro115.B30, Pro115.B31, Pro115.B32, Pro115.B33,Pro115.B34, Pro115.B35, Pro115.B36, Pro115.B37, Pro115.B38,Pro115.B39, Pro115.B40, Pro115.B41, Pro115.B42, Pro115.B43,Pro115.B44, Pro115.B45, Pro115.B46, Pro115.B47, Pro115.B48,Pro115.B49, Pro115.B50, Pro115.B51, Pro115.B52, Pro115.B53,Pro115.B54, Pro115.B55, Pro115.B56, Pro115.B57, Pro115.B58,Pro115.B59, Pro115.B60, Pro115.B61, Pro115.B62, Pro115.B63,Pro115.B64, Pro115.B65, Pro115.B66, Pro115.B67, Pro115.B68,Pro115.B69, Pro115.D1, Pro115.D2, Pro115.D3, Pro115.D4, Pro115.D5,Pro115.D6, Pro115.D7, Pro115.D8, Pro115.D9, Pro115.D10, Pro115.D11,Pro115.D12, Pro115.D13, Pro115.F2 and Pro115.F3.

[0290] If the MAb has been cloned, it will get the nomenclature"X.1," e.g., the first clone of Pro115.B7 will be referred to asB7.1, the second clone of B7 will be referred to as B7.2, etc. Forthe purposes of this invention, a reference to Pro115.B7 or B7 willinclude all clones, e.g., B7.1, B7.2, etc.

Immunogens and Antigens (Recombinant Proteins, HA & His Tags& Transfected Cells)

[0291] For the Constructs described below, nucleic acid moleculesencoding regions of Pro115 were inserted into various expressionvectors to produce recombinant proteins.

[0292] For purposes of illustration, the amino acid sequenceencoded by each construct is also included. However, the constructsmay include naturally occurring variants (e.g. allelic variants,SNPs) within the Pro115 region as isolated by the primers. Thesevariant sequences, and antibodies which bind to them are consideredpart of the invention as described herein.

Pro115 Construct 1 Sequence and Protein Production

[0293] A nucleic acid molecule encoding the full length Pro115protein, amino acids Met1 to Gly492, was inserted into a pCMV5His3vector at the PmeI/NheI sites. The vector comprises a sequence atthe 3' end of the cloning site encoding two transitional aminoacids, Ala and Ser, and a 10 His tag. The resulting vector with theinserted Pro115 nucleic acid fragment encodes a recombinant Pro115fusion protein with the 10 His-tag fused to the C-terminus of thePro115 protein. This recombinant plasmid encoding the full lengthPro115 His-tagged protein is herein referred to as "Pro115Construct 1". A representative amino acid sequence encoded byPro115 Construct 1 is presented in SEQ ID NO:1.

TABLE-US-00004 (SEQ ID NO: 1) Pro115 Construct 1 Amino AcidSequence 1 11 21 31 41 51 | | | | | | 1 MALNSGSPPA IGPYYENHGYQPENPYPAQP TVVPTVYEVH PAQYYPSPVP QYAPRVLTQA 61 SNPVVCTQPKSPSGTVCTSK TKKALCITLT LGTFLVGAAL AAGLLWKFMG SKCSNSGIEC 121DSSGTCINPS NWCDCVSHCP GGEDENRCVR LYGPNFILQV YSSQRKSWHP VCQDDWNENY181 GRAACRDMGY KNNFYSSQGI VDDSGSTSFM KLNTSAGNVD IYKKLYHSDACSSKAVVSLR 241 CIACGVNLNS SRQSRIVGGE SALPGAWPWQ VSLHVQNVHVCGGSIITPEW IVTAAHCVEK 301 PLNNPWHWTA FAGILRQSFM FYGAGYQVEKVISHPNYDSK TKNNDIALMK LQKPLTFNDL 361 VKPVCLPNPG MMLQPEQLCWISGWGATEEK GKTSEVLNAA KVLLIETQRC NSRYVYDNLI 421 TPAMICAGFLQGNVDSCQGD SGGPLVTSKN NIWWLIGDTS WGSGCAKAYR PGVYGNVMVF 481TDWIYRQMRA DGASHHHHHH HHHH

[0294] Pro115 protein expressed by Construct 1 was column purifiedusing standard techniques from cell culture of 293F cellstransfected with Construct 1. Samples from collected fractions weresubjected to SDS-PAGE and Western blot analysis for assessing thepurity of the protein. Purified fractions were pooled and dialyzedagainst PBS, pH 7.4. Pro115 expressed by Construct 1 was enzymaticactive.

Pro115 Construct 2 Sequence and Protein Production

[0295] A nucleic acid molecule encoding a mutant extra-cellularregion of Pro115 protein, amino acids Gly110 to Gly492, wasinserted into a modified pTT3 vector at the Pmel/Nhel sites. Thecodon encoding amino acid S441 was modified to encode A441,producing Pro115 mutant without protease activity.

[0296] The vector comprises a sequence at the 5' end of the cloningsite encoding an amino acid secretion signal sequence from humanstanniocalcin 1 (STC) and a sequence at the 3' end of the cloningsite encoding two transitional amino acids, Ala and Ser, and a 10His tag. The resulting vector with the inserted Pro115 nucleic acidfragment encodes a recombinant Pro115 fusion protein with aN-terminus STC secretion signal and a 10 His-tag fused to theC-terminus of the Pro115 protein. This recombinant plasmid encodingthe mutant extra-cellular region His-tagged protein is hereinreferred to as "Pro115 Construct 2". A representative amino acidsequence encoded by Pro115 Construct 2 is presented in SEQ IDNO:2.

TABLE-US-00005 Pro115 Construct 2 Amino Acid Sequence (SEQ ID NO:2) MLQNSAVLLVLVISASADIGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDAGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADGASHHHHHHHHHH

[0297] Pro115 protein expressed by Construct 2 was column purifiedusing standard techniques from mammalian cell culture transfectedwith Construct 2. Samples from collected fractions were subjectedto SDS-PAGE and Western blot analysis for assessing the purity ofthe protein. Purified fractions were pooled and dialyzed againstPBS, pH 7.4. Pro115 expressed by Construct 2 was not enzymaticactive.

Pro115 Construct 3 Sequence and Protein Production

[0298] A nucleic acid molecule encoding the protease domain ofPro115 protein, amino acids Ile236 to Gly492, was inserted into amodified pTT3 vector at the Pmel/Nhel sites. The vector comprises asequence at the 5' end of the cloning site encoding an amino acidsecretion signal sequence from human stanniocalcin 1 (STC) and asequence at the 3' end of the cloning site encoding twotransitional amino acids, Ala and Ser, and a 10 His tag. Theresulting vector with the inserted Pro115 nucleic acid fragmentencodes a recombinant Pro115 fusion protein with a N-terminus STCsecretion signal and a 10 His-tag fused to the C-terminus of thePro115 protein. This recombinant plasmid encoding the His-taggedPro115 protease domain protein is herein referred to as "Pro115Construct 3". A representative amino acid sequence encoded byPro115 Construct 3 is presented in SEQ ID NO:3.

TABLE-US-00006 Pro115 Construct 3 Amino Acid Sequence (SEQ ID NO:3) MLQNSAVLLVLVISASADIIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADGASHHHHHHHHHH

[0299] Pro115 protein expressed by Construct 3 was column purifiedusing standard techniques from mammalian cell culture transfectedwith Construct 3. Samples from collected fractions were subjectedto SDS-PAGE and Western blot analysis for assessing the purity ofthe protein. Purified fractions were pooled and dialyzed againstPBS, pH 7.4. Pro115 expressed by Construct 3 was enzymaticactive.

Pro115 Membrane Prep From RK3E-Pro115 Cells

[0300] For cell membrane preps, RK3E cells were transfected toexpress full length Pro115 using standard techniques. Approximately3 mL packed RK3E-Pro115 cells were homogenized with a Bellco Douncepestle in sucrose buffer and subjected to discontinuous gradientultracentrifugation (25000 rpm for 2 hours) in a Beckman SW28rotor. Membrane pellicules were collected, washed withphosphate-buffered saline, and centrifuged for 5 minutes at 2000rpm. The pellet containing the membrane fraction was left on iceovernight and was resuspended in 7.5 mL phosphate-buffered salineand frozen prior to content confirmation by western blotanalysis.

Pro115v1 Construct 1 Sequence and Protein Production

[0301] A splice variant of Pro115 named Pro115v1 has been describedin PCT/US2005/035607. A nucleic acid molecule encoding theextra-cellular region of Pro115v1, amino acids Gly110 to Leu257,was inserted into a modified pCMV5His3 vector at the PmeI/NheIsites.

[0302] The vector comprises a sequence at the 5' end of the cloningsite encoding an amino acid secretion signal sequence from humanstanniocalcin 1 (STC) linked to a 10 His tag. The resulting vectorwith the inserted Pro115v1 nucleic acid fragment encodes arecombinant Pro115v1 fusion protein with a N-terminus STC secretionsignal and a 10 His-tag. This recombinant plasmid encoding theHis-tagged Pro115v1 extra-cellular domain protein is hereinreferred to as "Pro115v1 Construct 1". A representative amino acidsequence encoded by Pro115 Construct 3 is presented in SEQ IDNO:4.

TABLE-US-00007 Pro115v1 Construct 1 Amino Acid Sequence (SEQ ID NO:4) MLQNSAVLLVLVISASADIHHHHHHHHHHGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCGESALTLGRDSSAHLGDSSRVQGPLGDWAWRASSTLTHDVIESLLQAEPWGSERLCFRPNLTQQVGDDRATEDCVIGTTRALNCHRKSVKMSKLFIKLEMQARNGGSCL

[0303] Pro115v1 protein expressed by Pro115v1 Construct 1 expressedusing standard techniques from mammalian cell culture (293F cells)transfected with Pro115v1 Construct 1. GnHCl denaturingpurification, followed by refolding, produced a native ornear-native protein. Samples from collected fractions weresubjected to SDS-PAGE and Western blot analysis for assessing thepurity of the protein. Purified fractions were pooled and dialyzedagainst PBS, pH 7.4.

Pro104 Construct 1 Sequence and Protein Production

[0304] Pro104 has previously been described in WO 99/60162 and WO00/16805. A nucleic acid molecule encoding a region of Pro104protein, amino acids Arg19-Trp297 was inserted into a modifiedpTJR3 vector. The vector comprises a sequence at the 5' end of thecloning site encoding an amino acid secretion signal sequence fromhuman stanniocalcin 1 (STC) and a sequence at the 3' end of thecloning site encoding two transitional amino acids, Ala and Ser,and a 10 His tag. The resulting vector with the inserted Pro104nucleic acid fragment encodes a recombinant Pro104 fusion proteinwith a N-terminus STC secretion signal and a 10 His-tag fused tothe C-terminus of the Pro104 protein. This recombinant plasmidencoding the protein is herein referred to as "Pro104 Construct 1".A representative amino acid sequence encoded by Pro104 Construct 1is presented in SEQ ID NO:5.

TABLE-US-00008 Pro104 Construct 1 Amino Acid Sequence (SEQ ID NO:5) MLQNSAVLLVLVISASATHEAEQSRKPESQEAAPLSGPCGRRVITSRIVGGEDAELGRWPWQGSLRLWDSHVCGVSLLSHRWALTAAHCFETYSDLSDPSGWMVQFGQLTSMPSFWSLQAYYTRYFVSNIYLSPRYLGNSPYDIALVKLSAPVTYTKHIQPICLQASTFEFENRTDCWVTGWGYIKEDEALPSPHTLQEVQVAIINNSMCNHLFLKYSFRKDIFGDMVCAGNAQGGKDACFGDSCCPLACNKNGLWYQIGVVSWGVGCGRPNRPGVYTNISHHFEWIQKLMAQSGMSQPDPSWASHHHHHHHHHH

[0305] Pro104 protein expressed by Pro104 Construct 1 was columnpurified using standard techniques from mammalian cell culturetransfected with Construct 1. Samples from collected fractions weresubjected to SDS-PAGE and Western blot analysis for assessing thepurity of the protein. Purified fractions were pooled and dialyzedagainst PBS, pH 7.4.

Cln101 Construct 1 Sequence and Protein Production

[0306] Cln101 has previously been described in WO 05/021709. Anucleic acid molecule encoding a region of Cln101 protein, aminoacids Asp23-Pro158 was inserted into a modified pCMV5His2 vector.The vector comprises a sequence at the 5' end of the cloning siteencoding an amino acid secretion signal sequence from humanstanniocalcin 1 (STC) and a sequence at the 3' end of the cloningsite encoding two transitional amino acids, Ala and Ser, and a 10His tag. The resulting vector with the inserted Cln101 nucleic acidfragment encodes a recombinant Cln101 fusion protein with aN-terminus STC secretion signal and a 10 His-tag fused to theC-terminus of the Cln101 protein. This recombinant plasmid encodingthe protein is herein referred to as "Cln101 Construct 1". Arepresentative amino acid sequence encoded by Cln101 Construct 1 ispresented in SEQ ID NO:6.

TABLE-US-00009 Cln101 Construct 1 Amino Acid Sequence (SEQ ID NO:6) MLQNSAVLLVLVISASATHEAEQDIIMRPSCAPCWFYHKSNCYGYFRKLRNWSDAELECQSYGNGAHLASILSLKEASTIAEYISCYQRSQPIWIGLHDPQKRQQWQWIDGAMYLYRSWSGKSMGGNKHCAEMSSNNNFLTWSSNECNKRQHFLCKYRPASHHHHHHHHHH

[0307] Cln101 protein expressed by Cln101 Construct 1 was columnpurified using standard techniques from mammalian cell culturetransfected with Construct 1. Samples from collected fractions weresubjected to SDS-PAGE and Western blot analysis for assessing thepurity of the protein. Purified fractions were pooled and dialyzedagainst PBS, pH 7.4.

Cln242 Construct 1 Sequence and Protein Production

[0308] Cln242 has previously been described in WO 01/92528 and isalso known as Cln165. A nucleic acid molecule encoding a region ofCln242 protein, amino acids Met1-Asp198 was inserted into amodified pCMV5His2 vector. The vector comprises a sequence at the3' end of the cloning site encoding two transitional amino acids,Ala and Ser, and a 10 His tag. The resulting vector with theinserted Cln242 nucleic acid fragment encodes a recombinant Cln242fusion protein with a 10 His-tag fused to the C-terminus of theCln242 protein. This recombinant plasmid encoding the protein isherein referred to as "Cln242 Construct 1". A representative aminoacid sequence encoded by Cln242 Construct 1 is presented in SEQ IDNO:7.

TABLE-US-00010 Cln242 Construct 1 Amino Acid Sequence (SEQ ID NO:7) MSGGHQLQLAALWPWLLMATLQAGFGRTGLVLAAAVESERSAEQKAIIRVIPLKMDPTGKLNLTLEGVFAGVAEITPAEGKLMQSHPLYLCNASDDDNLEPGFISIVKLESPRRAPRPCLSLASKARMAGERGASAVLFDITEDRAAAEQLQQPLGLTWPVVLIWGNDAEKLMEFVYKNQKAHVRIELKEPPAWPDYDDI ASHHHHHHHHHH

[0309] Cln242 protein expressed by Cln242 Construct 1 was purifiedusing standard techniques from mammalian cell culture transfectedwith Construct 1. Samples from collected fractions were subjectedto SDS-PAGE and Western blot analysis for assessing the purity ofthe protein. Purified fractions were pooled and dialyzed againstPBS, pH 7.4.

Ovr110 Construct 1 Sequence and Protein Production

[0310] A nucleic acid molecule encoding Ovr110 (also known as B7H4)protein was inserted into a vector for expression. The resultingvector with the inserted Ovr110 nucleic acid fragment encodes arecombinant Ovr110 fusion protein with a 10 His-tag fused to theC-terminus of the Ovr110 protein. This recombinant plasmid encodingthe protein is herein referred to as "Ovr110 Construct 1". Arepresentative amino acid sequence encoded by Ovr110 Construct 1 ispresented in SEQ ID NO:8.

TABLE-US-00011 Ovr110 Construct 1 Amino Acid Sequence (SEQ ID NO:8) MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLKASHHHHHHHHHH

[0311] Ovr110 protein expressed by Ovr110 Construct 1 was purifiedusing standard techniques from mammalian cell culture transfectedwith Construct 1. Samples from collected fractions were subjectedto SDS-PAGE and Western blot analysis for assessing the purity ofthe protein. Purified fractions were pooled and dialyzed againstPBS, pH 7.4.

Immunizations

[0312] Mice were immunized with various Pro115 and Pro115v1constructs to generate anti-Pro115 MAbs capable of binding toPro115 in bodily fluids and on a cell surface. MAbs are capable ofbinding to Pro115 expressing cells, killing Pro115 expressingcells, and have utility as in-vivo therapeutic agents, and in-vivoand in-vitro diagnostic agents.

[0313] For the A-series MAb fusion, mice were immunized with theprotein encoded by Pro115 Construct 1 (described above). Theprotein was expressed in mammalian cells.

[0314] For the B-series MAb fusion, mice were immunized with theprotein encoded by Pro115 Construct 2 (described above). Theprotein was expressed in mammalian cells.

[0315] For the D-series MAb fusion, mice were immunized with theprotein encoded by Pro115 Construct 3 (described above). Theprotein was expressed in mammalian cells.

[0316] For the F-series MAb fusion, mice were immunized withmembranes isolated from Pro115-transfected RK3E cells (describedabove).

[0317] For each series, groups of 8 BALB/c mice were immunizedintradermally in both rear footpads. All injections were 25 uL perfoot. For the A-, B-, and D-series, the first injection of 10 ug ofantigen per mouse was in Dulbecco's phosphate buffered saline(DPBS) mixed in equal volume to volume ratio with Titermax goldadjuvant (Sigma). Subsequently, mice were immunized twice weeklyfor 5 weeks. For the 2.sup.nd through 9.sup.th injection, mice wereimmunized with 10 ug of antigen in 20 uL of DPBS plus 5 uL ofAdju-phos adjuvant (Accurate Chemical & Scientific Corp.,Westbury, N.Y.) per mouse. The final immunization consisted of 10ug antigen diluted in DPBS alone. For the F-series, mice wereinjected with membranes prepared from RK3E cells stably transfectedwith Pro115. Mice were immunized twice weekly for five weeks with10 ug RK3E-Pro115 membranes in 25 uL PBS per foot.

Hybridoma Fusion

[0318] Four days after the final immunization, mice were sacrificedand draining lymph node (popliteal and inguinal) tissue wascollected by sterile dissection. Lymph node cells were dispersedusing a Tenbroeck tissue grinder (Wheaton #347426, VWR, Brisbane,Calif.) followed by pressing through a sterile sieve (VWR) intoDMEM and removing T-cells via anti-CD90 (Thy1.2) coated magneticbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany).

[0319] These primary B-cell enriched lymph node cells were thenimmortalized by electro-cell fusion (BTX, San Diego, Calif.) withthe continuous myeloma cell line P3x63Ag8.653 (Kearney, J. F. etal., J. Immunology 123: 1548-1550, 1979). The myeloma and B-cellswere pooled at a 1:1 ratio for the fusion. These fusion cultureswere distributed at 2 million cells per plate into wells of 96 wellculture plates (Costar #3585, VWR). Successfully fused cells wereselected by culturing in selection medium (DMEM/15% FBS) containing2.85 .mu.M Azaserine, 50 .mu.M Hypoxanthine (HA) (Sigma) or 50.mu.M Hypoxanthine, 0.2 .mu.M Aminopterin, 8 .mu.M Thymidine (HAT)(Sigma) supplemented with recombinant human IL-6 (Sigma) at 0.5ng/mL. Cultures were transitioned into medium (DMEM/10% FBS/IL-6)without selection for continued expansion and antibodyproduction.

[0320] Alternatively, fused cells were cultured in bulk inselection medium for 6 days and then plated, 1 cell per well, into96 well culture plates by cell sorting (Coulter Elite). Cells werecultured and transitioned into medium (DMEM/10% FBS/IL-6) withoutselection for continued expansion and antibody production.Hybridomas generated by this plating method are designated A101.1to A122.1 in Table 1.

[0321] Supernatants from wells were screened by enzyme linked solidphase immunoassay (ELISA) and flow cytometry for antibodies bindingto recombinant Pro115 protein and to Pro115 expressed on cells.Monoclonal cultures, consisting of the genetically uniform progenyfrom single cells, were established after the screening procedure,by sorting of single viable cells into wells of two 96 well plates,using flow cytometry (Coulter Elite; Beckman-Coulter, Miami, Fla.).The resulting murine B-cell hybridoma cultures were expanded usingstandard tissue culture techniques. Selected hybridomas werecryopreserved in fetal bovine serum (FBS) with 10% DMSO and storedin Liquid Nitrogen at -196.degree. C. to assure maintenance ofviable clone cultures.

ELISA Screening & Selection of Hybridomas Producing Pro115Specific Antibodies

A Series Antibodies

[0322] Hybridoma cell lines were selected for production of Pro115specific antibodies by direct ELISA. Wells were coated withproteins encoded by Pro115 Construct 1, Pro115v1 Construct 1,Pro104 Construct 1 (negative control) or Cln101 Construct 1(negative control); all described above. To coat wells one ug/mLprotein in PBS (100 uL/well) was incubated overnight in 96 wellpolystyrene EIA plates (Costar #9018, VWR) at 4.degree. C. Theplate wells were washed twice with Tris buffered saline with 0.05%Tween20, pH 7.4 (TBST). Nonspecific binding capacity was blocked byfilling the wells (300 ul/well) with TBST/0.5% bovine serum albumin(TBST/BSA) and incubating for >30 minutes at room temperature(RT). The wells were emptied and filled with 50 uL/well TBST/BSA toprevent them from drying out during the sample collection process.Hybridoma culture medium sample was added to the wells (50 uL) andincubated for 1 hour at RT. The wells were washed 3 times withTBST. One hundred uL of alkaline phosphatase conjugated goatanti-mouse IgG (Fc) with minimal cross-reactivity to human Fc(PN115-055-071, Jackson Immunoresearch), diluted 1:5000 in1BST/BSA, was added to each well and incubated for >1 hour atRT. The wells were washed 3 times with TBST. One hundred uL ofalkaline phosphatase substrate para-nitrophenylphosphate (pNPP)(Sigma) at 1 mg/mL in 1 M Diethanolamine buffer pH 8.9 (Pierce) wasadded to each well and incubated for 20 min at RT. The enzymaticreaction was quantified by measuring the solution's absorbance at405 nm wavelength.

TABLE-US-00012 TABLE 1 ELISA with supernatants of A-serieshybridomas. ELISA OD [405 nm] Hybridoma Pro115 Pro115v1 Pro104Cln101 A1 1.3067 3.3473 0.1028 0.1189 A2 0.8973 0.4626 0.09650.1020 A3 1.0153 0.1163 0.1041 0.1032 A4 1.7338 0.1473 0.13050.1378 A5 2.0018 4.0000 0.1040 0.1150 A6 1.0826 0.0964 0.09900.1052 A7 1.3652 0.0997 0.1102 0.1168 A8 1.7950 1.8729 0.11210.1077 A9 1.3678 0.9320 0.1346 0.1012 A10 0.9950 0.3955 0.10550.1210 A11 0.7001 0.1144 0.1172 0.1232 A12 0.9987 0.1529 0.09730.1152 A13 0.9895 0.7933 0.1024 0.1075 A14 1.2906 0.1014 0.10620.1030 A15 1.1788 0.0962 0.2407 0.1038 A16 1.4956 0.3009 0.10480.1135 A17 1.4809 3.4570 0.1118 0.1252 A18 1.1674 0.0940 0.09620.1177 A19 1.2368 0.0984 0.0959 0.1038 A20 0.7900 0.1078 0.09820.1144 A21 0.6521 0.1075 0.1059 0.1111 A22 1.0836 0.1039 0.11710.1090 A23 0.7594 0.1016 0.1053 0.1162 A25 2.0331 0.1059 0.13260.1051 A24 2.2609 3.5445 0.1013 0.1040 A101.1 1.0194 0.1302 0.10570.1139 A102.1 1.2091 0.1129 0.0998 0.1221 A103.1 1.0498 0.09890.1065 0.1164 A104.1 1.2561 0.1042 0.1062 0.1191 A106.1 1.15050.0990 0.1012 0.1009 A107.1 1.5703 4.0000 0.1194 0.1042 A108.11.0811 2.3565 0.1053 0.1076

[0323] Supernatants from 32 hybridomas produced an absorbance valueof greater than 0.5 in wells coated with Pro115 Construct 1 andless than 0.16 in wells coated with Cln101 Construct 1, indicatingpreferential antibody binding to Pro115. Antibodies from 19 of the32 hybridomas did not bind to Pro115v1 Construct1 and Pro104Construct 1, indicating specific binding to Pro115. Thesehybridomas are designated A3, A4, A6, A7, A11, A12, A14, A18, A19,A20, A21, A22, A23, A25, A101, A102, A103, A104, and A106.Antibodies from 12 of the 32 hybridomas bound to Pro115 Construct 1and Pro115v1 Construct 1, indicating binding to the common domainof Pro115 and Pro115v1 (amino acids Met1 to Cys148). Thesehybridomas are designated A1, A2, A5, A8, A9, A10, A13, A16, A17,A24, A107, and A108. One antibody, named A15, bound to Pro115Construct 1, and has some cross-reactivity with Pro104 Construct 1.Selected hybridomas were expanded for further analysis.

B Series Antibodies

[0324] Hybridoma cell lines were selected for production of Pro115specific antibody by direct ELISA and Cell ELISA. The direct ELISA,which was performed as described above, binding to proteins encodedby Pro115 Construct 2, and Cln242 Construct1 (negative control) wasevaluated.

[0325] For the Cell ELISA, the binding of antibodies to RK3E cellsstably transduced with either Pro115 (RK3E-Pro115-SA) or alkalinephosphatase (RK3E-AP; negative control) was evaluated. 25,000 cellsin 100 ul growth medium were plated per well of a 96-well platecoated with Poly-D-Lysine (#15600, Pierce). Cells were incubatedovernight, and 50 ul hybridoma supernatant or purified antibody (1ug/ml final concentration) were added to each well. Cells wereincubated on ice for 30 min. Wells were emptied and washed withTBST/BSA. Cells were then fixed for 10 min on ice by adding 100 ul4% formaldehyde in TBS. Wells were emptied and washed withTBST/BSA. 300 ul TBST/BSA was added to each well. After incubatingcells for 30 min at RT, wells were emptied and washed twice withTBST/BSA. 100 ul biotin-conjugated rabbit(Fab2) anti-mouse IgG(P/N315-066-046; Jackson Immunoresearch, West Grove, Pa.), diluted1:20,000 in TBST/BS, were added per well to stain the cells. After30 min incubation at RT, wells were emptied and washed twice withTBST/BSA. 100 ul Streptavidin-HRP conjugate (#21126; Pierce),diluted 1:20,000 in TBST/BSA, were added to each well and cellswere incubated for 30 min at RT. Wells were washed twice withTBST/BSA. 100 ul of HRP substrate 3,3',5,5'-tetramethyl benzidine(#S1599; Dako Cytomation, Carpinteria, Calif.) were added. Thereaction was stopped by adding 100 ul 1N hydrochloric acid, usuallyafter 20 min or when the desired staining intensity was reached.The enzymatic reaction was quantified by measuring the solution'sabsorbance at 450 nm wavelength.

[0326] Evaluation of supernatants from hybridomas in direct ELISAand cell ELISA cells is shown in Table 2. Values in the last column(column 6) are the ratio of OD values from cells transduced withPro115 (column 4) to cells transduced with alkaline phosphatase(column 5).

TABLE-US-00013 TABLE 2 Cell ELISA OD Direct ELISA OD [450 nm] [405nm] RK3E- Pro115 Cln242 Pro115 RK3E-AP Pro115:AP Clone # Construct2 Construct 1 cells cells ratio B1 1.3064 0.1508 0.9892 0.2944 3.4B2 0.7695 0.1315 1.0996 0.3332 3.3 B3 1.0286 0.1251 0.9038 0.38592.3 B4 1.0292 0.0908 0.6268 0.3935 1.6 B5 0.8952 0.0893 0.88090.4520 1.9 B6 1.1827 0.0847 1.0687 0.4424 2.4 B7 0.9649 0.09031.0307 0.4748 2.2 B8 0.6000 0.0849 1.1025 0.3132 3.5 B9 1.34980.1311 0.8373 0.3217 2.6 B10 0.5779 0.1131 0.3590 0.2972 1.2 B110.8952 0.1255 1.0869 0.3686 2.9 B12 0.7692 0.1487 0.4794 0.4600 1.0B13 0.5551 0.1328 0.5080 0.4029 1.3 B14 1.3539 0.1087 0.9859 0.37702.6 B15 0.7326 0.1419 0.4957 0.3501 1.4 816 0.6789 0.1089 0.46450.2897 1.6 B17 1.2082 0.0858 0.7619 0.2888 2.6 B18 1.1609 0.09900.6447 0.3016 2.1 B19 1.0495 1.4265 0.3724 0.2681 1.4 B20 0.59220.0755 0.9259 0.3893 2.4 B21 1.0629 0.0909 0.6376 0.3631 1.8 B220.5780 0.0901 0.4196 0.3111 1.3 B23 0.6406 0.0765 0.8860 0.3273 2.7B24 1.0601 0.0756 0.4474 0.3196 1.4 B25 1.4708 0.0788 0.9597 0.34432.8 B26 0.8612 0.0725 0.5162 0.4455 1.2 B27 0.6300 0.0712 0.39330.3862 1.0 B28 1.0939 0.0707 0.6197 0.4396 1.4 B29 0.9180 0.08160.8671 0.2934 3.0 B30 1.0810 0.0957 0.7064 0.6884 1.0 B31 0.53890.0792 0.8135 0.3848 2.1 B32 0.7776 0.0707 0.6028 0.4344 1.4 B331.0041 0.1537 0.4214 0.2723 1.5 B34 0.7255 0.1361 0.9487 0.3022 3.1B35 0.0897 0.1220 0.3343 0.2767 1.2 B36 0.2022 0.1527 0.4554 0.41951.1 B37 0.1477 0.1679 0.3871 0.3589 1.1 B38 0.1586 0.1247 0.39530.2979 1.3 B39 0.4560 0.1201 0.4370 0.3923 1.1 B40 0.3565 0.09430.3109 0.2885 1.1 B41 0.3313 0.0722 0.3843 0.3030 1.3 B42 0.20890.0728 0.4389 0.4574 1.0 B43 0.1879 0.0738 0.4412 0.4390 1.0 B440.1416 0.0801 0.5787 0.6334 0.9 B45 0.0881 0.0833 0.5593 0.5082 1.1B46 0.0715 0.0780 0.4392 0.3773 1.2 B47 0.1023 0.0811 0.5300 0.52521.0 B48 0.4945 0.0722 0.5469 0.4100 1.3 B49 0.3490 0.0764 0.44620.5483 0.8 B50 0.1172 0.0710 0.3423 0.2479 1.4 B51 0.4495 0.06961.1329 0.5829 1.9 B52 0.2492 0.0670 0.3861 0.2762 1.4 B53 0.27040.0755 0.9000 0.3024 3.0 B54 0.0939 0.0761 0.3913 0.3289 1.2 B550.3086 0.0889 0.4052 0.3179 1.3 B56 0.3672 0.0762 0.5163 0.2300 2.2B57 0.2947 0.1389 0.3990 0.3367 1.2 B58 0.3650 0.1274 0.3170 0.24811.3 B59 0.4041 0.1361 0.7632 0.2126 3.6 B60 0.0845 0.1451 0.45190.3941 1.1 B61 0.0806 0.1198 0.4669 0.3454 1.4 B62 0.1221 0.10810.3660 0.3019 1.2 B63 0.4344 0.0835 0.3918 0.3671 1.1 B64 0.20890.1211 0.3694 0.2681 1.4 B65 0.2957 0.1547 0.4746 0.5397 0.9 B660.2248 0.0995 0.3960 0.3803 1.0 B67 0.1017 0.1376 0.3143 0.2592 1.2B68 0.3047 0.0746 0.3950 0.3117 1.3 B69 0.2215 0.0769 0.3823 0.32221.2

[0327] Selected hybridomas were expanded for further analysis.

D Series Antibodies

[0328] Hybridoma cell lines were selected for production of Pro115specific antibody by direct ELISA as described above. Binding toproteins encoded by Pro115 Construct 2, and Cln242 Construct1(negative control) was evaluated. Supernatants from hybridomas thatproduced an absorbance value of greater than 1.50 in wells coatedwith Pro115 Construct and less than 0.15 in wells coated withCln242 Construct1 (negative control), indicated specific binding toPro115. All thirteen specific hybridomas, named Pro115.D1 toPro115.D13, were expanded and cryopreserved.

F Series Antibodies

[0329] Hybridoma cell lines were selected for production of Pro115specific antibody by direct ELISA as described above. Supernatantsfrom 2 hybridomas produced an absorbance value of greater than 0.5in wells coated with Pro115 Construct 2 and less than 0.1 in wellscoated with an irrelevant protein, indicating preferential antibodybinding to Pro115. These hybridomas are designated F2 and F3.

Flow Cytometry Screening for Cell Surface Binding of Pro115MAbs

[0330] Selected hybridoma supernatants were analyzed by flowcytometry for cell surface staining of transfected 293F cells andtumor cell lines.

[0331] 293F cells were transiently transfected with expressionplasmids for Pro115 Construct 1, Pro115v1 Construct 1, Pro104Construct 1, and Ovr110 Construct 1 using 293fectin (Invitrogen) astransfection reagent. 48 hours post-transfection, cells were washedonce with 10 ml Ca.sup.+2/Mg.sup.+2free DPBS and then 7 ml of warm(37.degree. C.) Cellstripper (Mediatech, Herndon, Va.) was addedper 150 cm.sup.2 flask. The cells were then incubated for 5 minutesat 37.degree. C. with tapping of the flask to remove tightlyattached cells. The cells were removed and pipetted several timesto break aggregates, then immediately placed in DMEM/10% FBS/5 mMsodium butyrate. The cells were then centrifuged down for 5 minutesat 1300 rpm and resuspended in DMEM/10% FBS/5 mM sodium butyrate.The cells were incubated at 37.degree. C. for a 30 minute recoveryperiod. Prior to staining, viability of the cells was measuredusing Guava Viacount (Guava Cytometers, Foster City, Calif.) andcultures with >90% viability were selected for staining withMAbs.

[0332] Cells from cultures selected for MAb staining were aliquotedat 0.5-1.0.times.10.sup.6 cells/well in 96-well v-bottom plates(VWR) and centrifuged for 2 minutes at 1500 rpm. Supernatants wereaspirated and plates briefly shaken on a vortex mixer to resuspendthe cells, then 200 ul of DPBS/3% FBS/0.01% Na Azide (FACS buffer)was added to each well. Centrifugation and aspiration was repeated,then 25 uL of sequential dilutions of hybridoma supernatant orpurified MAb was added to the cells. Plates were stored on ice for15 min., then washed and centrifuged as above, in 200 uL of FACSbuffer. This washing procedure was repeated twice and then 25 uL ofphycoerythrin (PE) conjugated donkey anti-mouse IgG Fc antibody(Jackson Immunoresearch Laboratories) were added to cells. After 15minutes on ice the cells were washed twice, as above and thenresuspended in 250 uL of FACS buffer for analysis on an Elitefluorescent activated cell sorter (FACS) (Beckman-Coulter).

TABLE-US-00014 TABLE 3 Cell surface staining of transfected 293Fcells with hybridoma supernatants 293- 293- 293- 293- Pro115Pro115v1 Pro104 Ovr110 % % % % cells cells cells cells posi- posi-posi- posi- tive MFI tive MFI tive MFI tive MFI A1 96.2 7.490 64.80.691 22.8 0.344 A2 37.3 0.558 6.7 0.304 1.9 0.233 A3 27.8 0.4556.4 0.315 1.3 0.236 A4 48.7 0.614 1.4 0.259 1.6 0.221 A5 97.812.500 15.3 0.369 1.0 0.216 A6 23.9 0.435 2.3 0.266 0.9 0.211 A737.8 0.552 93.2 2.220 86.3 1.100 A8 98.7 10.500 61.1 0.643 38.50.406 A9 42.1 0.627 3.2 0.269 1.5 0.223 A10 45.0 0.875 1.1 0.2570.7 0.208 A11 62.2 0.809 1.2 0.260 0.9 0.216 A12 91.2 2.060 2.10.264 1.1 0.221 A13 39.7 0.580 3.1 0.280 1.6 0.229 A14 16.4 0.3822.4 0.262 1.7 0.219 A15 2.1 0.275 2.0 0.271 0.7 0.218 A16 11.90.349 1.3 0.257 0.5 0.214 A17 97.8 9.850 16.1 0.371 1.4 0.237 A1859.0 0.721 4.8 0.298 1.8 0.230 A19 19.2 0.403 0.7 0.244 0.7 0.214A20 46.1 0.637 85.2 2.690 86.5 2.530 A21 28.5 0.503 2.6 0.270 0.70.218 A22 64.8 0.837 8.6 0.311 2.1 0.237 A23 69.9 1.010 2.6 0.2660.9 0.219 A24 95.7 6.840 2.7 0.267 0.6 0.210 A25 50.1 0.631 2.50.266 1.9 0.222 A101.1 43.5 0.653 3.2 0.279 1.2 0.223 A107.1 76.33.17 10.5 0.363 1.3 0.302 A108.1 41.2 0.791 5.1 0.286 2.1 0.230 B223.6 5.81 2.2 1.57 80.4 3.58 70.2 2.26 B3 67.2 3.68 1.5 0.48 1.50.332 2.5 0.334 B5 87.6 3.96 3.6 1.73 80.8 2.43 74.1 1.93 B7 59.93.72 1.7 0.439 0.7 0.314 1.2 0.305 B8 19.9 5.75 1.1 0.422 1 0.3171.1 0.308 B20 22.7 5.82 1.2 0.526 10.2 0.136 8.8 0.379 B22 97.112.8 63.6 7.71 97.1 5.15 91.4 3.41 B23 22.2 5.9 1.1 0.465 1.9 0.3372.6 0.34 B29 22.9 5.49 1.4 0.438 0.9 0.305 1.5 0.313 B31 33.9 4.811.2 0.429 0.9 0.313 1.2 0.313 B34 36.5 4.62 1.4 0.471 1.1 0.344 1.70.321 B48 76.7 4.06 1.2 0.45 1.3 0.334 1.6 0.324 B53 22.2 6.6 1.50.617 16.4 1.01 12.9 0.456 B56 92.8 5.01 1.9 1.5 80.1 1.7 68.7 1.33B64 98.8 23.8 97 23.6 99.7 28.1 94.9 6.01 D1 11 0.388 4.8 0.353 D234.9 0.627 0.6 0.326 D3 32.9 0.575 0.3 0.317 D4 10.4 0.397 2 0.35D5 29.4 0.585 3.8 0.273 D6 44 0.756 0.3 0.315 D7 34.8 0.591 1.40.342 D8 0.8 0.313 0.2 0.32 D9 5.4 0.369 0.3 0.331 D10 18 0.416 0.10.3 D11 1.5 0.302 0.4 0.312 D12 19.5 0.436 0.2 0.306 D13 31.3 0.5560.2 0.327

[0333] Hybridomas producing anti-Pro115 MAbs that boundspecifically to cells expressing Pro115 at the cell surface but notto negative control cells with surface protein Ovr110 were furtheranalyzed by flow cytometry for cell surface staining of tumor celllines LNCaP and HCT116.

Culture and Stimulation of Cancer Cell Lines

[0334] LNCaP cells natively express Pro115 and expression levelscan be further upregulated by addition of the hormone mibolerone tothe culture medium. HCT116 cells do not express Pro115 and servedas negative control. Using standard cell culture techniques andmethods cancer cell lines LNCaP (CRL-1740; ATCC, Manassas, Va.) andHCT-116 (CCL-247; ATCC) were cultured in RPMI/10% FBS and DMEM/10%FBS, respectively. LNCaP cells were stimulated for 48 hours byadding Mibolerone (Perkin Elmer Life and Analytical Science,Wellesley, Mass.) to the culture medium to a final concentration of10 nM. Anti-Pro115, anti-Ricin (negative control) and anti-CD71(positive control) antibodies were evaluated for binding toun-stimulated and stimulated LNCaP cells and HCT-116 cells. Percentpositive cells and the MFI for each cell antibody and cell type arelisted in Table 4 below.

TABLE-US-00015 TABLE 4 Cell surface staining of cancer cell lineswith hybridoma supernatants. Stimulated Unstimulated LNCaP LNCaPHCT116 % Cells % Cells % Cells Sample Positive MFI Positive MFIPositive MFI Anti-Ricin 3.8 0.463 0.9 0.262 2.6 0.306 Anti-CD7119.7 0.958 98.1 7.17 81.8 2.58 B3 35.5 1.65 45 0.878 1.2 0.289 B732.9 1.56 38.2 0.776 0.5 0.271 B8 24.3 1.14 28.2 0.668 0.7 0.273B20 30.2 1.44 39.2 0.787 1.7 0.312 B23 31.4 1.48 42.9 0.834 1.20.292 B29 31.1 1.46 39.2 0.802 1.2 0.288 B31 49.8 2.22 79.8 1.53 10.291 B34 55.3 2.56 84.2 1.69 1.2 0.296 B48 44.9 2.03 70.4 1.3 4.40.34 B53 38 1.79 57.2 1.05 1.2 0.295 D1 34.4 0.766 1.6 0.377 20.80.494 D2 74.6 1.9 3.4 0.351 2.7 0.285 D3 62.7 1.3 3.6 0.328 1.20.218 D4 56.3 1.1 2.8 0.355 2.9 0.289 D5 5.3 0.461 8.2 0.335 4.60.266 D6 35.4 0.797 0.3 0.306 2 0.246 D7 63.9 1.34 7.3 0.43 11.60.368 D8 6.5 0.49 0.7 0.268 1.1 0.247 D9 38.4 0.883 3.3 0.307 2.20.266 D10 43.6 0.949 0.9 0.282 0.1 0.194 D11 30.9 0.776 0.7 0.277 10.231 D12 43 0.927 0.6 0.289 0.5 0.251 D13 78.6 1.82 9 0.444 35.10.512 F2 69.4 1.250 0.1 0.275 F3 49.9 0.766 0.3 0.282

The results in Table 4 above demonstrate that all tested B and Fseries antibodies specifically bind to Pro115-positive LNCaP cells,but not to Pro115-negative HCT116 cells. Cell surface binding canbe enhanced by stimulating LNCaP cells with mibolerone, whichupregulates Pro115 expression. Antibodies D1 and D15 stained HCT116cells indicating that these antibodies cross-reacted with a proteinother than Pro115. The other D series antibodies specifically boundto stimulated LNCaP cells.

Cloning of Hybridomas Producing Pro115 Specific MAb

[0335] Based on data from the ELISA and flow cytometry experimentsabove, the following hybridomas were selected for single cellcloning into 96 well culture plates by cell sorting (CoulterElite): A1, A4, A5, A8, A12, A107.1, B3, B7, B20, B23, B29, B31,B34, B53, D2, D3, D5, D7, D9, D10, D11, D12, F2 and F3.

[0336] After 2 weeks of culture, supernatants from subclonedhybridomas were tested by direct ELISA on wells coated with Pro115Construct 1. Up to 3 positive subclones per parent hybridoma wereexpanded and cryopreserved in liquid nitrogen. MAbs were purifiedfrom overgrown hybridoma supernatants and analyzed by flowcytometry for cell surface binding of Pro115-transfected 293F cellsas well as binding to tumor cell lines. Flow Cytometry bindingresults are displayed below in Tables 5 and 6.

TABLE-US-00016 TABLE 5 Cell surface staining of Pro115 transfected293F cells with purified MAbs. Pro115-Transfected 293F CellsControl 293F Cells Mean Mean % Cells Fluorescence % CellsFluorescence Sample Positive Intensity Positive IntensityAnti-Ricin 0.9 0.425 0.9 0.324 Anti-CD71 18.6 5.6 99.7 21 A1.1 864.27 1.5 0.34 A4.1 9.2 0.695 0.7 0.316 A5.1 88.2 4.97 1.9 0.329A8.1 82.8 4.29 54.7 1.95 A12.1 3.7 0.569 20.6 1.05 A17.1 85.3 7.3823.0 0.83 A22.1 34.2 0.96 67.2 2.99 A24.1 28.4 0.83 8.4 0.52A107.1.1 92.4 10.90 31.0 0.95 B3.1 96.6 11.20 56.3 1.96 B7.1 93.49.99 2.5 0.51 B20.1 78.7 5.83 0.0 0.30 B23.1 71.3 4.70 0.0 0.25B29.1 77.4 5.54 0.1 0.31 B31.1 70.6 4.46 0.2 0.39 B34.1 80.0 5.671.3 0.45 B53.1 68.2 4.15 0.0 0.29

TABLE-US-00017 TABLE 6 Cell surface staining of cancer cell lineswith purified MAbs. Stimulated Unstimulated LNCaP LNCaP HCT116 %Cells % Cells % Cells Sample Positive MFI Positive MFI Positive MFIAnti-Ricin 3.8 0.463 0.9 0.262 2.6 0.306 Anti-CD71 19.7 0.958 98.17.17 81.8 2.58 A1.1 92.0 5.160 7.6 0.386 2.3 0.295 A4.1 15.4 0.6063.0 0.301 A5.1 92.0 5.170 17.6 0.534 2.6 0.300 A8.1 89.9 4.520 7.70.346 A12.1 35.1 0.947 4.1 0.318 A17.1 73.7 2.420 37.2 1.32 3.60.307 A22.1 45.4 1.220 32.0 1.21 88.8 2.110 A24.1 69.9 2.570 24.91.12 5.4 0.341 A107.1.1 88.9 4.490 31.7 1.26 4.1 0.312 B3.1 99.727.900 86.2 3.32 1.3 0.271 B7.1 99.7 28.200 16.9 1.00 1.4 0.274B20.1 99.3 25.800 16.2 0.95 1.7 0.278 B23.1 99.3 25.200 7.3 0.801.3 0.268 B29.1 99.4 27.500 22.8 1.09 1.6 0.276 B31.1 99.2 24.10015.8 0.97 1.7 0.275 B34.1 99.7 26.500 30.2 1.23 1.8 0.271 B53.199.2 24.100 6.1 0.77 1.1 0.261 D2.1 70.8 2.710 1.9 0.351 14.8 0.425D3.1 70.9 2.460 4.6 0.438 25.8 0.512 D5.1 39.3 1.060 6.7 0.406 62.60.964 D7.1 65.8 2.010 2.4 0.331 51.9 0.744 D9.1 71.8 2.500 0.30.287 36.9 0.611 D10.1 52.0 1.510 0 0.254 2.0 0.289 D11.1 52.01.470 0 0.252 2.5 0.285 D12.1 17.0 0.643 0 0.254 2.2 0.287 F2.199.7 26.0 2.6 0.312 F3.11 99.3 32.0 6.6 0.365

[0337] MAbs A22.1, D2.1, D3.1, D5.1, D7.1 and D9.1 boundnonspecifically to Pro115-negative cells. All other MAbs of the A-,B-, D- and F series listed in Tables 5 and 6 bound specifically toPro115-expressing cells demonstrating that the subcloned hybridomasrobustly produced anti-Pro115 specific MAbs.

Pro115 MAb Checkerboard ELISA

[0338] A checkerboard ELISA was ran with Pro115 A and B seriesMabs. High binding polystyrene plates (Corning Life Sciences) werecoated overnight at 4.degree. C. with 0.2 .mu.g/well of a firstanti-Pro115 MAb. The coating solution was aspirated off and freebinding sites were blocked with 300 .mu.l/well Superblock-TBS(Pierce Biotechnology, Illinois) on a shaker for 1 hour at roomtemperature (RT). After washing 4 times with washing buffer(TBS+0.05% Tween20), 100 .mu.l of Pro115 Construct 2 protein at 2.mu.g/ml (in Assay Buffer (TBS, 1% BSA, 1% Mouse Serum, 1% CalfSerum, and 0.1% ProClin.TM.) was added to each well. The plate wasincubated for 60 minutes on a shaker. For detection, 100 .mu.l of asecond biotinylated MAb (0.2 .mu.g/ml) was added to each well andincubated for 1 hour at room temperature (RT), while shaking. Afterwashing, 100 .mu.L of Streptavidin-HRP conjugate (Jackson Lab) at1:80,000 dilution in TBS, was added to each well. Plates were thenincubated with shaking at RT for 30 min. After washing the plate,100 uL/well of TMB-Stable Stop substrate (Moss, Inc.) was added toeach well and the plate was incubated at RT, covered and on theshaker for 15 minutes. The reaction was stopped using 100.mu.l/well 1N HCl, and the plates were read at 450 nm using aSpectramax 190 plate reader (Molecular Devices).

[0339] The results of the checkerboard ELISA are shown in Tablexxx. Capturing MAbs are listed on the Y-axis with detecting MAbs onthe X-axis. Each antibody was tested as both a coating anddetecting antibody, in all possible combinations. OD numbers at 450nm are shown. Similar pairing patterns of antibodies indicate thatthese antibodies bind to similar epitopes. The anti-Pro115 MAbsshown in Table 7 detect at least six distinct epitopes. AntibodiesA1.1, A107.1, A5.3 and A8.1 (group 1) recognize a similar epitope.Antibodies B20.1, B23.1, B29.1, B31.1 and B34.1 (group 2) recognizea similar epitope which is different the epitope recognized bygroup 1. Antibodies A4.1, A25.1 and A102.1 (group 3) recognize asimilar epitope which is distinct from the epitopes recognized bygroups 1 and 2. Antibodies B3.1 and B7.1 recognize a similarepitope which is different from the epitope recognized by groups 1,2, and 3. Antibodies A12.1 and B53.1 are unique. Their pairingpatterns do not resemble the pairing pattern of any other antibodyindicating that they bind to epitopes distinct from the epitopesrecognized by the other antibodies. For formatting convenience, theantibody nomenclature of Table 7 below has been simplified to showthe parent name only (e.g. A4) and not the full clone name (e.g.A4.1).

TABLE-US-00018 TABLE 7 Checkerboard ELISA with selected Pro115 MAbscoat 1.degree. detecting 2.degree. MAb Mab A1 A107 A5 A8 B23 B31B29 B34 B20 B53 A4 A102 A25 A12 B7 B3 A1 0.15 0.2 0.37 0.29 3.722.97 4 3.99 4 1.67 2.54 1.12 2.05 0.8 4 4 A5 0.09 0.14 0.18 0.163.95 3.05 4 4 4 2.52 2.44 0.92 1.89 0.78 4 4 A8 0.26 0.28 0.56 0.483.95 3.29 4 4 4 2.03 4 1.54 2.68 1.14 4 4 A107 0.05 0.1 0.08 0.072.69 1.56 3.27 3.01 3.92 1.4 1.45 0.59 1.07 0.48 3.96 4 B53 1.231.36 2.37 2.65 3.82 2.99 3.92 3.34 4 0.12 0.49 0.21 0.38 0.21 3.984 A4 3.12 2.63 3.94 4 2.39 1.89 3.76 3.49 3.86 1.14 0.37 0.16 0.371.09 4 4 A25 3.37 3.02 3.97 4 1.83 1.19 2.76 3.06 3.62 0.89 0.620.24 0.36 0.99 4 4 A102 1.02 0.97 2.02 1.64 0.38 0.23 0.77 0.770.98 0.31 0.19 0.07 0.13 0.25 2.09 2.55 B31 2.6 2.79 3.42 4 0.270.17 0.77 0.98 1.04 2.39 1.03 0.42 0.85 0.42 3.83 3.99 B29 2.873.13 3.71 4 0.1 0.09 0.2 0.25 0.28 3.91 0.96 0.37 0.87 0.61 3.93 4B34 3.51 3.54 3.88 4 0.1 0.12 0.22 0.26 0.28 4 1.28 0.45 0.91 0.614 4 B20 3.96 4 4 4 0.14 0.13 0.37 0.46 0.56 4 1.49 0.56 1.11 0.83 44 B23 4 4 4 4 0.2 0.16 0.7 0.73 0.96 4 3.03 0.98 2.02 1.46 4 4 A122 1.95 2.78 2.94 1.43 0.99 2.35 2.66 2.99 0.48 1.5 0.57 0.94 0.142.05 2.55 B3 3.15 3.7 4 4 3.92 3.19 4 4 4 3.99 1.74 0.63 1.1 0.290.3 0.32 B7 4 3.93 4 4 4 4 4 4 4 4 3.02 1.16 1.89 0.54 0.621.02

Pro115 MAb Competition ELISA

[0340] A competition ELISA was ran with selected Pro115 Mabs. Highbinding polystyrene plates (Corning Life Sciences) were coatedovernight at 4.degree. C. with 0.2 .mu.g/well monoclonal antibodyagainst a hexa-Histidine tag. The coating solution was aspiratedoff and free binding sites were blocked with 300 .mu.l/wellTBST/BSA for 1 hour. After washing 3 times with TBST buffer, 100.mu.l of Pro115 Construct 2 protein at 2 .mu.g/ml (in TBST/BSA) wasadded to each well. The plate was incubated for 60 minutes on ashaker and washed 3 times with TBST buffer. One-hundred .mu.l of afirst, unlabeled Pro115 MAb (20 .mu.g/ml in TBST/BSA) was added toeach well, followed by incubation for 30 minutes. Forty .mu.l of asecond, biotinylated Pro115 MAb (7 .mu.g/ml in TBST/BSA) was addedto each well, followed by incubation for 30 minutes. The plate waswashed and 100 .mu.l of Streptavidin-AP conjugate (Jackson Lab) at1:2,000 dilution in TBST/BSA was added to each well. Plates werethen incubated for 30 min. After washing the plate, 100 uL/well ofpNPP substrate was added to each well, the plate was incubated for60 minutes and read at 405 nm.

[0341] The results of the competition ELISA are shown in Table 8.Unlabeled competition MAbs are listed on the Y-axis withbiotinylated detecting MAbs on the X-axis. Each antibody was testedas both a competing and detecting antibody, in all possiblecombinations. Numbers shown are OD values for a pair of competitionand detecting MAbs normalized to the OD value of the same detectingMAb in combination with an irrelevant competition MAb which doesnot bind to Pro115. A ratio of 1.0 indicates that the competitionMAb does not block the binding of the detecting MAb to Pro115; bothMAbs bind to different epitopes. A ratio close to 0.0 indicatesthat the competition MAb blocks the binding of the detecting MAb toPro115; both MAbs bind to similar or identical epitopes. AntibodiesA5.3 and A8.1 block each other and both are blocked by A4.1,indicating that they bind to similar or identical epitopes.Antibodies B34.1 and F2.1 block each other and are notsignificantly blocked by other antibodies, indicating that theybind to similar or identical epitopes. Antibodies A24.1, B53.1,D11.1, D3.1, A17.1, D2.1, B7.1 and F3.11.1 have unique inhibitionpatterns suggesting that each antibody binds to a distinct epitope.Binding of F3.11.1 is not inhibited by any other antibody,indicating that its epitope is distant from epitopes recognized byother Pro115 MAbs.

TABLE-US-00019 TABLE 8 Competition ELISA with selected Pro115 MAbs.comp detecting Mab Mab A4.1 A24.1 B53.1 D11.1 D3.1 A17.1 A5.3 A8.1D2.1 F3.11.1 F2.1 B34.1 B7.1 A4.1 0.82 0.22 0.44 0.44 0.39 0.150.11 0.01 0.46 0.89 0.95 0.48 0.31 A5.3 0.88 0.26 0.42 0.31 0.370.1 0.01 0 0.4 0.87 0.96 0.66 0.44 A8.1 1.17 0.51 0.66 0.69 0.510.23 0.04 0 0.59 0.86 0.96 0.77 0.65 A17.1 1.04 0.67 0.99 0.59 0.35-0.02 0.83 0.88 0.92 1.01 0.94 0.95 0.77 D3.1 0.71 0.57 0.45 0.060.03 0.45 0.71 0.71 1 0.75 0.99 0.71 0.6 D11.1 0.91 0.65 1.2 0.10.74 0.82 0.82 0.84 1.26 0.94 1.08 1.15 0.91 D2.1 0.66 0.48 0.691.12 0.56 0.35 0.65 0.67 0.02 0.78 0.97 0.78 0.69 A24.1 0.76 0.040.92 0.83 0.33 0.47 0.76 0.71 0.86 0.79 0.95 0.99 0.84 B7.1 1.260.8 1.64 1.35 0.53 0.66 1.01 0.7 1.06 1.33 0.96 1.01 0.02 B3.1 0.890.98 2.01 1.67 1.17 0.47 0.81 0.67 1.21 1.46 0.86 0.96 0.03 B34.10.86 0.54 1.48 1.25 0.75 0.49 0.64 0.93 0.75 1.54 0.05 0.01 0.74F2.1 0.93 0.91 1.94 1.74 1.07 0.97 0.92 0.95 1.24 1.77 0.14 0.041.06 B53.1 0.94 0.59 0.02 1.07 0.89 0.59 0.8 0.83 0.88 1.04 0.960.8 0.77 F3.11.1 1.12 0.74 1.77 1.07 0.83 0.79 0.86 0.82 1.11 0.10.98 0.98 0.97

Off-Ranking Analysis of Pro115 mAbs

[0342] Dissociation rate constants (kd) were calculated fromsurface plasmon resonance measurements using a BIACORE 3000instrument (BiaCore, Piscataway, N.J.). An anti-Mouse Ig surfacewas used to capture each antibody, followed by an injection of theprotein encoded by Pro115 Construct 2 over the capturedantibody.

[0343] Flow cell 1 of a CM5 sensor chip (BiaCore) was used as acontrol surface for reference subtractions, and was activated andderivatized with nonspecific rabbit IgG using standard methods.Flow cells 2, 3, and 4 were derivatized with anti-Mouse IgG dilutedto 35 ug/mL in 10 mM acetate as suggested. Standard amine coupling(BiaCore) was used to immobilize approximately 12000 RU of ligandper flow cell. Purified antibodies were diluted in HBS-EP runningbuffer (BiaCore) to 20 ug/mL and passed over flow cells 2, 3, or 4at 15 uL/min flow rate, 3 minute injection. Following mouse Igloading, 31 ug/mL Pro115 Construct 2 protein was injected at 10uL/minute for 3.4 minutes. The dissociation time was 5 minutes. Theregeneration of the chip surface, or removal of captured hybridomasupernatants binding to the antigen between cycles, was performedby injecting 10 mM glycine pH 2.0 for 20 seconds and 10 mM glycinepH 1.5 for 10 seconds at 100 uL/minute.

[0344] The sensor chip derivatization procedure was performed byusing the BiaCore's surface preparation and binding wizard includedin the BiaCore control software. The off-ranking results presentedin Table 9a below were automatically fitted using the separateka/kd function included in the BiaCore analysis software, assuminga 1:1 Langmuir binding model.

TABLE-US-00020 TABLE 9a Pro115 mAb kinetics Mab kd Mab RU AntigenRU A1.1 2.55E-04 625.3 124.5 A4.1 1.31E-04 459.9 141.2 A5.11.96E-04 433.4 130.6 A8.1 3.36E-04 957.3 157.9 A22.1 1.91E-04 821.2202.7 A25.1 1.31E-04 401.3 121.9 A107.1.1 1.69E-04 681.5 145.5A123.1.1 6.27E-04 378.6 117.6 B7.1 2.26E-04 314.4 153.2 B20.12.39E-04 414.6 225.4 B23.1 7.24E-04 312.6 164.8 B29.1 1.73E-04321.8 180 B31.1 1.16E-03 449.5 216.2 B34.1 3.05E-04 414.4 242.9B53.1 4.05E-04 211.8 120.1 D2.1 4.07E-04 524.8 169.9 D3.1 2.27E-04601.4 152.4 D5.1 2.49E-04 389.6 122.2 F2.1 3.24E-04 422.5 207.9F3.11.1 3.41E-04 554.5 223.7

These results demonstrate that anti-Pro115 antibodies are useful asdiagnostic or therapeutic agents. Pro115 MAbs in table 9a have kdvalues and binding characteristics (described herein) whichdemonstrate their utility as diagnostic agents.

Affinity Measurements of Pro115 Antibodies

[0345] ELISA plates were coated with 0.3 ug anti-His antibodyHis.A6.1 (in 100 ul PBS) and blocked with 300 ul TBST/BSA.Twenty-five ng of Pro115 Construct2 protein in 100 ul TBST/BSA wereadded to each well and plates were incubated for 1 hour. Plateswere washed 3 times with 300 ul TBST. One-hundred ul ofbiotinylated Pro115 antibody was added to each well. Theconcentration of the antibody ranged from 1 uM to 0.5 nM. Plateswere incubated for 3 hours and washed 3 times with 300 ul TBST.One-hundred ul Streptavidin-AP conjugate (1:2000 dilution inTBST/BSA; Jackson Immunoresearch) was added to each well. Plateswere incubated for 30 min and washed 3 times with 300 ul TBST. Thealkaline phosphatase signal was developed by addition of pNPPsubstrate and quantified by OD measurement at 405 nm. OD signalsgenerated by a negative control antibody were subtracted from ODsignals generated by Pro115 antibodies at equivalent antibodyconcentrations. Binding curves (net OD values plotted against theantibody concentration) were analyzed with the software Prism(GraphPad). The dissociation constant KD was calculated bynonlinear regression assuming a one-site binding model. Table 9bbelow list the KD in nanomolar concentration of Pro115antibodies.

TABLE-US-00021 TABLE 9b Pro115 antibody affinities mAb KD [nM] B7.10.11 B34.1 0.23 B53.1 1428 F2.1 0.19 F3.11.1 603

These results demonstrate that anti-Pro115 antibodies are useful asdiagnostic or therapeutic agents. An antibody with nanomolar rangeaffinity is considered useful as a therapeutic agent. Pro115 MAbsB7.1, B34.1, F2.1 and F3.11.1 have KD values and bindingcharacteristics (live cell binding) which demonstrate their utilityas therapeutic agents.

Pro115 MAb Isotypes

[0346] The isotypes of the anti-Pro115 MAbs were determined usingcommercially available mouse monoclonal antibody isotypingimmunoassay test kits (IsoStrip, Roche Diagnostic Corp.,Indianapolis, Ind.). Results of the isotyping are listed in Table10.

TABLE-US-00022 TABLE 10 Pro115 MAb Isotypes Clone Isotype A4.1 IgG1kappa A5.1 IgG1 kappa A12.1 IgG2a kappa A107.1 IgG1 kappa B7.1 IgG1kappa B34.1 IgG1 kappa

Western Blots

[0347] Protein extracts for western blot analysis were preparedfrom transfected RK3E-Pro115 and control RK3E-AP (alkalinephosphatase) cells as well as from different cancer cell linesColo-205 (CCL-222, ATCC), T84 (CLL-248; ATCC), LNCaP and HCT-116.Colo-205, T84 and LNCaP are Pro115 positive, and HCT-116 is Pro115negative as judged by quantitative PCR analysis. Cells wereincubated for 10 min in Cell Lysis Buffer (50 mM Tris, pH7.5, 50 mMNaCl, 2 mM EDTA, 1% NP-40; complete protease inhibitors (Roche)were added freshly). The lysate was centrifuged for 10 min at4.degree. C. at 10,000 g and the protein concentration of thecleared supernatant was determined (BCA Assay, #23227; Pierce).Fifteen ug total proteins were separated by electrophoresis onNuPAGE 4-12% Bis-Tris gels (Invitrogen) under denaturing conditionsin Novex-XCell II Minicell gel apparatus (Invitrogen) andsubsequently transferred to PVDF membranes using an XCell II BlotModule (Invitrogen Life Technologies). Following the transfer ofproteins, the membranes were blocked in TBST with 5% non fat milkat room temperature for at least an hour, followed by incubationovernight at 4.degree. C. with primary antibody (hybridomasupernatant or purified MAb at 2 ug/mL), and then withhorseradish-peroxidase conjugated goat anti-mouse IgG secondaryantibody (Jackson Immunoresearch Laboratories, Inc.) for one hour.The membranes were visualized by chemiluminescence using an ECLadvance western blotting detection kit (Amersham Biosiences,Piscataway, N.J.). The HA antibody was from obtained from Covance(Berkeley, Calif.). Results of western blot experiments withanti-Pro115 A-series MAbs and D-series MAbs are summarized inTables 11 and 12. Band intensity is categorized as weak (-),intermediate (+/-), strong (+), or not detected (ND). The size ofthe band(s) detected is also indicated.

TABLE-US-00023 TABLE 11 Pro115 A-series MAb Western Blot Results.Cell line RK3E-Pro115HA RK3E-AP Colo-205 T84 Bands Bands BandsBands MAb detected Intensity detected Intensity Detected Intensitydetected Intensity Anti-HA 54 kDa +/- none 28 kDa +/- A1.1 54 kDa +none 54 kDa +/- 54 kDa +/- 38 kDa - 38 kDa + 38 kDa + A4.1 54 kDa+/- none 54 kDa - 54 kDa - 28 kDa +/- 28 kDa - 28 kDa - A5.1 54 kDa+ none 54 kDa +/- 54 kDa +/- 38 kDa +/- 38 kDa + 38 kDa + 16 kDa+/- 16 kDa - 16 kDa - A8.1 54 kDa +/- none 54 kDa - 54 kDa - 38 kDa- 38 kDa +/- 38 kDa +/- 16 kDa - A12.1 54 kDa + none 54 kDa +/- 54kDa +/- 38 kDa +/- 38 kDa + 38 kDa + 16 kDa +/- 16 kDa - 16 kDa -A107.1.1 54 kDa + none 38 kDa +/- 16 kDa +/-

TABLE-US-00024 TABLE 12 Pro115 D-series MAb Western Blot Results.Cell line LNCaP HCT116 Bands Bands MAb detected Intensity detectedIntensity D2.1 54 kDa + none 38 kDa +/- 28 kDa + D3.1 54 kDa +/-none 28 kDa +/- D5.1 54 kDa + none 28 kDa +

[0348] Due to proteolytic cleavage of Pro115 three distinct bandsare observed. The 54 kDa band represents the full length protein,the 38 kDa band represents the N-terminal product of the cleavedprotein which has the LDL and SRCR domains, and the 28 kDa bandrepresents the C-terminal product of the cleaved protein which hasthe protease domain. The antibodies A1.1, A4.1, A5.1, A8.1, A12.1,A107.1, D2.1, D3.1, and D5.1 specifically recognized Pro115 inwestern blots. Antibodies A1.1, A5.1, A8.1, A12.1, and A107.1recognize full-length Pro115 and the N-terminal LDL/SRCR domain.Antibodies A4.1, D2.1, D3.1, and D5.1 recognize full-length Pro115and the C-terminal protease domain. Some Pro115 antibodiesincluding those from the B-series, F2 and F3 did not bind to Pro115in western blot experiments, but did bind Pro115 in surfacestaining ELISAs above indicating these antibodies bind to anon-linear or conformational epitope destroyed in the denaturationof the western blot experiment.

[0349] Western Blots of Human Tissue

[0350] Western blots were conducted as described above usingdiseased and normal human tissues. Prostate and colonadenocarcinoma (ACA) tissues samples were evaluated alongsidenormal (NRM) prostate and colon tissues. Results of theseexperiments with anti-Pro115 MAb A4.1 are summarized in Tables 13below. Band intensity is categorized as weak (-), intermediate(+/-), strong (+), or not detected (ND). The size of the band(s)detected is also indicated.

TABLE-US-00025 TABLE 13 Western Blot of Pro115 in human cancertissues Primary Human Tissue Samples Pro ACA #1 Pro ACA #2 Pro NRMCIn ACA CIn NRM Bands Bands Bands Bands Bands detected Intensitydetected Intensity detected Intensity detected Intensity detectedIntensity 54 kDa +/- 54 kDa +/- 54 kDa - 54 kDa ND 54 kDa ND 28 kDa+ 28 kDa + 28 kDa + 28 kDa +/- 28 kDa +/-

[0351] These results demonstrate that Pro115 is expressed in higherlevels in prostate and colon tumors than in normal prostate andcolon tissue.

Example 2

Epitope Mapping of Pro115

Pro115 MAb Peptide Mapping

[0352] The epitopes recognized by antibodies from the Pro115 A, B,D and F series were determined by screening overlapping peptidesfor reactivity with the antibodies through an ELISA-based assay.Thirty-nine overlapping peptides were ordered from SynPep (Dublin,Calif.). Peptides 1-38 were 15-mers overlapping 5 amino acids withthe adjacent peptides. Peptide 39 contained 7 amino acids. Thepeptide sequences started at amino acid W106 after thetransmembrane domain and ended at G429 at the C-terminus of thePro115 protein. These peptides span the extracellular region of themature Pro115 protein. The peptides were provided in solution witha range of 1-3 mg/ml DMSO. A 1:400 dilution was made in PBS of eachpeptide and 50 .mu.l were added to each well in duplicate on96-well 4.times. Costar plates (#3690) (Costar Corporation;Cambridge, Mass.) and left overnight. Pro115 Construct 2 describedabove was used as a positive control on each 96-well plate. Thenext day, the plates were flicked dry and blocked with TBST 0.5%BSA for approximately 1 hour. Anti-Pro115 antibodies (50 .mu.l)were added at 20 pg/ml per well and incubated at room temperaturefor approximately 2 hours. The plates were washed 3 times with TBSTwash buffer. The secondary conjugate, goat anti-mouse Ig Fc-AP,(Pierce, Rockford, Ill.) was diluted 1:5000 in a TBST/BSA solutionand 50 .mu.l was added to each well. The plates were shaken for 2hours at room temperature. The plates were washed 3 times before 50.mu.l of substrate was added to each well and incubated for 15minutes at room temperature. The substrate used was pNPP in1.times.DEA (1 mg/ml). To visualize the assay, plates were read at405 nm on a SpectraMaxPlus plate reader (Molecular Devices,Sunnyvale, Calif.).

[0353] Table 14 below outlines the results of the anti-Pro115antibody peptide binding experiments described above. Anti-Pro115 Aand D series antibodies showed strong specific reactivity withPro115 peptides. MAbs A1.1, A5.1, A8.1, A17.1, and A107.1 bound topeptides which cover the LDL receptor class A domain (C113-C148;peptides 1, 2, 3, 4 and 5). Anti-Pro115 antibodies which bind tothe LDL receptor domain of Pro115 inhibit function of the LDLreceptor domain. MAb A12.1 bound to peptide 12, which is in theSRCR domain (V149-N241; peptides 5, 6, 7, 8, 9, 10, 11, 12, 13, 14and 15). Anti-Pro115 antibodies which bind to the SRCR domain ofPro115 inhibit function of the SRCR domain. MAbs A17.1, A24.1,A102.1, A107.1, D3.1, D5.1, D7.1 and D12.1 bound to peptides whichare part of the protease domain (I256-Q487; peptides 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39).Anti-Pro115 antibodies which bind to the protease domain of Pro115inhibit function of the protease domain. MAb A24.1, bound topeptide 34, which contains amino acid Ser441, the catalytic centerof the protease domain. Anti-Pro115 antibodies which bind topeptides or epitopes containing amino acids which are part of theserine protease catalytic triad (His296, Asp345 or Ser441) inhibitprotease activity of Pro115.

[0354] The following antibodies did not map to any peptide: A4.1,A25.1, A123.1, B3.1, B7.1, B20.1, B23.1, B29.1, B31.1, B34.1, D9.1,D10.1, D11.1, F2.1, F3.11.1. Since these antibodies bind torecombinant Pro115 in an ELISA assay, they recognize either aconformational epitope or a region that includes apost-translational modification. Despite not recognizing a linearepitope of the peptides evaluated, these antibodies have utility inbinding Pro115 domains or inhibiting Pro115 biochemical andbiological functions.

[0355] Anti-Pro115 antibodies which inhibit the function oractivity of Pro115 domains inhibit or reduce Pro115 biochemicalfunctions such as LDL receptor binding, scavenger receptor (SRCRdomain) binding, protease activity or protease autocleavage andactivation.

TABLE-US-00026 TABLE 14 Peptide mapping of Abs. Abs which generatedan absorbance of 0.4 or higher in an ELISA assay are shown.Absorbance of irrelevant control antibodies was 0.1 OD. Abs bindingto Peptide SEQ Strong Moderate Peptide Peptide ID Binding BindingNumber Sequence NO OD > 1.0 1.0 > OD > 0.4 1WKFMGSKCSNSGIEC 9 A8.1 2 SGIECDSSGTCINPS 10 A5.1, A1.1, A22.1 A8.1,A107.1 3 CINPSNWCDGVSHCP 11 A17.1 A22.1 4 VSHCPGGEDENRCVR 12 A17.1A22.1 5 NRCVRLYGPNFILQM 13 A22.1 6 FILQMYSSQRKSWHP 14 A22.1 7KSWHPVCQDDWNENY 15 A22.1 8 WNENYGRAACRDMGY 16 A22.1 9RDMGYKNNFYSSQGI 17 A12.1 A22.1 10 SSQGIVDDSGSTSFM 18 11STSFMKLNTSAGNVD 19 12 AGNVDIYKKLYHSDA 20 13 YHSDACSSKAVVSLR 21 14VVSLRCIACGVNLNS 22 15 VNLNSSRQSRIVGGE 23 D3.1 16 IVGGESALPGAWPWQ 24A22.1 17 AWPWQVSLHVQNVHV 25 18 QNVHVCGGSIITPEW 26 A22.1 19ITPEWIVTAAHCVEK 27 20 HCVEKPLNNPWHWTA 28 21 WHWTAFAGILRQSFM 29 22RQSFMFYGAGYQVEK 30 23 YQVEKVISHPNYDSK 31 D3.1 24 NYDSKTKNNDIALMK 32A22.1 25 IALMKLQKPLTFNDL 33 A102.1, D5.1 26 IALMKLQKPLTFNDL 34 27LPNPGMMLQPEQLCW 35 D12.1 A22.1 28 EQLCWISGWGATEEK 36 A22.1 29ATEEKGKTSEVLNAA 37 A22.1 30 VLNAAKVLLIETQRC 38 A107.1 A22.1, D2.131 ETQRCNSRYVYDNLI 39 D3.1, A22.1 D7.1 32 YDNLITPAMICAGFL 40 33CAGFLQGNVDSCQGD 41 A17.1 A22.1 34 SCQGDSGGPLVTSKN 42 A24.1 35VTSKNNIWWLIGDTS 43 A22.1 A8.1, B53.1 36 IGDTSWGSGCAKAYR 44 37AKAYRPGVYGNVMVF 45 A22.1 A8.1 38 NVMVFTDWIYRQMRA 46 39 RQMRADG 47D3.1

Pro115 and TMPRSS Family Sequence Alignment

[0356] FIG. 1 is an alignment of human Pro115 with other members ofthe TMPRSS family and with mouse Pro115. The Refseq accessionnumbers are: NP.sub.--005647.2 (TMPRSS2, human Pro115),NP.sub.--056590.2 (mouse TMPRSS2, mouse Pro115), NP.sub.--002142.1(TMPRSS1), NP.sub.--076927.1 (TMPRSS3), NP.sub.--063947.1(TMPRSS4), NP.sub.--110397.1 (TMPRSS5), NP.sub.--705837.1(TMPRSS6), XP.sub.--293599.6 (TMPRSS7), NP.sub.--892018.1(TMPRSS9), NP.sub.--006578.2 (TMPRSS10), NP.sub.--872365.1(TMPRSS12), NP.sub.--114435.1 (TMPRSS13), the disclosures of whichare herein incorporated by reference. The alignment was performedusing the publicly available ClustalW 1.83 software using thedefault setting. Identical amino acids in a given position amongthe aligned proteins are marked with "*", conserved substitutionsare marked with ":" and semi-conserved substitutions are markedwith ".". The LDL receptor class A domain is underlined by twosolid lines ( ) the scavenger receptor cysteine-rich domain isunderlined with a broken line (. . . .) the serine protease domainis underlined by a single solid line (______) The three amino acidscomprising the catalytic center are highlighted by a darkbackground.

[0357] Because mouse Pro115 would not be immunogenic in mice,differences between human Pro115 and mouse Pro115 are likelyresponsible for the immune response in mice immunized with Pro115polypeptides and generation of the anti-Pro115 antibodies describedherein. The overlapping peptides analyzed in conjunction with thecross species Pro115 alignment allowed us to determine with greateraccuracy the epitopes and specific residues recognized byanti-Pro115 antibodies. The alignment of human Pro115 with mousePro115 and other human members of the TMPRSS family demonstrateimmunogenic regions of Pro115 and specific epitopes and residuesthat antibodies which are specific for Pro115 bind to.

Pro115 MAb Epitope Mapping

[0358] Antibody A8.1 was strongly reactive with peptides 1 and 2,indicating that the common amino acid sequence SGIEC is recognizedby this antibody. Since this sequence and residues I118 and E119are specific to Pro115 among the human TMPRSS family members, A8.1recognizes this epitope or specific residues within it. AntibodyA17.1 was strongly reactive with peptides 3, 4 which share thecommon amino acid sequence VSHCP (SEQ ID NO:48). A17.1 was alsoreactive to peptide 33 which contains the epitope VDSCQ (SEQ IDNO:49) which has some homology to VSHCP (SEQ ID NO:48).Additionally, residue N433 is unique to Pro115 among the TMPRSSfamily members. Since sequences VDSCQ (SEQ ID NO:49) and VSHCP (SEQID NO:48) share homology and residues H138 and N433 are specific toPro115 among the human TMPRSS family members, A17.1 recognizesthese epitopes or specific residues within them.

[0359] Antibody A12.1 was strongly reactive with peptide 9. Anamino acid change in peptide 9 between the human and mousesequences occurs at position 186 where arginine (R) is replaced bylysine (K) (RDMG (SEQ ID NO:50) to KDMG (SEQ ID NO:51)).Additionally, residues D187, K191, N192, N193, F194, Y195 and Q198are unique to Pro115 among the TMPRSS family members. Since thissequence and residues are specific to Pro115 among the human TMPRSSfamily members, A12.1 recognizes this epitope or specific residueswithin it.

[0360] Antibody A5.1 was strongly reactive with peptide 2, but notoverlapping adjacent peptides 1 and 3, indicating that an aminoacid sequence unique to peptide 2 is recognized by this antibody.An amino acid change in this sequence between the human and mousesequences occurs at position 121 where aspartic acid (D) isreplaced by glutamine (G) (DSSG (SEQ ID NO:52) to GSSG (SEQ IDNO:53)). Since this sequence and residues D121 and T125 arespecific to Pro115 among the human TMPRSS family members, A5.1recognizes this epitope or specific residues within it.

[0361] Antibody D3.1 was strongly reactive with peptides 23, 31,and 39, and has lower reactivity towards peptide 15, but was notreactive to overlapping adjacent peptides. Amino acid changes inthese peptides between the human and mouse sequences occur atpositions 247, 248, 250, 251 326, 413, 415, 417, 491 and 492. D3.1may recognize an epitope generated by sequence YQVE (SEQ ID NO:54),NYDSK (SEQ ID NO:55), VYDNL (SEQ ID NO:56) or NLNSS (SEQ ID NO:57).Since these sequences and residues are specific to Pro115 among thehuman TMPRSS family members, D3.1 recognizes these epitopes orspecific residues within them.

[0362] Antibodies A102.1 and D5.1 were strongly reactive withpeptide 25 but not overlapping adjacent peptides 24 and 26,indicating that an amino acid sequence unique to peptide 25 isrecognized by this antibody. There are 2 amino acid changes inpeptide 25 between the human and mouse sequences. Positions 353 and356 are changed from lysine (K) and threonine (T) to threonine (T)and alanine (A), respectively (KPLT (SEQ ID NO:58) to TPLA (SEQ IDNO:59)). Since this sequence is specific to Pro115 among the humanTMPRSS family members, A102.1 and D5.1 recognizes this epitope orspecific residues within it.

[0363] Antibody D7.1 was strongly reactive with peptide 31 but notoverlapping adjacent peptides 30 and 32, indicating that an aminoacid sequence unique to peptide 31 is recognized by this antibody.There are 3 amino acid changes in peptide 31 between the human andmouse sequences. Positions 415, 417 and 419 are changed fromarginine (R), valine (V) and aspartic acid (D) to lysine (K),isoleucine (I) and aspartagine (N), respectively (RYVYD (SEQ IDNO:60) to KYIYN (SEQ ID NO:61)). Therefore, A5.1 recognizes thisepitope or specific residues within it.

[0364] Antibody D12.1 was strongly reactive with peptide 27 but notoverlapping adjacent peptides 26 and 28, indicating that an aminoacid sequence unique to peptide 27 is recognized by this antibody.In peptide 27, the sequence QPEQL (SEQ ID NO:62)(human Pro115) ischanged to DLDQE (SEQ ID NO:63) (mouse Pro115). Additionally,residues M371, M372, Q374, E376, Q377 and L378 are unique to Pro115among the TMPRSS family members. Since this sequence and residuesM371, M372, Q374, E376, Q377 and L378 are specific to Pro115 amongthe human TMPRSS family members, D12.1 recognizes this epitope orspecific residues within it.

[0365] Antibody A24.1 is strongly reactive with peptide 34 but notoverlapping adjacent peptides 33 and 35, indicating that an aminoacid sequence unique to peptide 34 is recognized by this antibody.An amino acid change in peptide 34 between the human and mousesequences occurs at position 448 where serine (S) is replaced byleucine (L) (VTSK (SEQ ID NO:64) to VTLK (SEQ ID NO:65)).Additionally, residues T447 and S448 are unique to Pro115 among theTMPRSS family members. Since this sequence and residues M371, M372,Q374, E376, Q377 and L378 are specific to Pro115 among the humanTMPRSS family members, A24.1 recognizes this epitope or specificresidues within it. Peptide 34 also contains residue S441 which ispart of the catalytic triad. Antibodies which bind to peptide 19,peptide 24 or peptide 34 which respectively contain residues H296,D345 and S441 of the catalytic triad are capable of acting asagonists or antagonists to modulate the enzymatic activity ofPro115.

[0366] Antibodies which bind the same epitope of Pro115 as theantibodies of the present invention or compete for binding of theepitope of Pro115 bound by the antibodies of present invention arespecific to Pro115. In addition, anti-Pro115 antibodies which bindto a linear or conformational epitope containing one or more ofresidues W106, M109, S111, N115, I118, E119, D121, T125, N131,W132, H138, G141, N146, Y152, P154, I157, M160, Q164, R165, K166,H169, Q173, N179, R182, A183, D187, K191, N192, N193, F194, Y195,Q198, D202, G205, S206, T207, M210, K211, L212, N213, T214, A216,G217, N218, V219, D220, I221, Y222, D229, A230, S232, K234, A235,I242, V246, N247, N249, R252, Q253, N277, P301, N303, W306, H307,L315, R316, F319, M320, F321, Y322, Y326, Q327, D338, N343, K362,M371, M372, Q374, E376, Q377, L378, K390, E395, A399, K401, L403,E406, L419, A423, N433, T447, S448, I452, W454, I456, D458, K467,Y469, R470, G475, M478, V479, T481, R486, R489, G492 of the Pro115protein are specific to Pro115. Residues N128, N213 and N249 areN-glycosylation sites on Pro115 and are unique to Pro115 among theTMPRSS family members.

Example 3

Pro115 Cell Surface Expression in LNCaP Cells

[0367] LNCaP cells were stimulated for 48 hours by addingMibolerone (10 nM final concentration) to the culture medium. Cellswere either directly harvested for the determination of the totalcellular Pro115 expression or the cell surface proteins werebiotinylated prior to the harvest for the determination of the cellsurface expression of Pro115.

[0368] To determine the total cellular Pro115 expression instimulated and unstimulated LNCaP cells, cells were incubated for10 min in Cell Lysis Buffer (50 mM Tris, pH7.5, 50 mM NaCl, 2 mMEDTA, 1% NP-40; complete protease inhibitors (Roche) were addedfreshly). The lysate was centrifuged for 10 min at 4.degree. C. at10,000 g then the protein concentration of the cleared supernatantwas determined (BCA Assay, #23227; Pierce). Fifteen ug totalproteins were separated by electrophoresis on NuPAGE 4-12% Bis-Trisgels (Invitrogen) under denaturing conditions in Novex-XCell IIMinicell gel apparatus (Invitrogen) and subsequently transferred toPVDF membranes using an XCell II Blot Module (Invitrogen LifeTechnologies). Following the transfer of proteins, the membraneswere blocked in TBST with 5% non fat milk at room temperature forat least an hour, followed by incubation overnight at 4.degree. C.with purified primary MAb Pro115.A12.1 at a concentration of 2ug/mL, and then with horseradish-peroxidase conjugated goatanti-mouse IgG secondary antibody (Jackson ImmunoresearchLaboratories, Inc.) and finally visualized by chemiluminescenceusing an ECL advance western blotting detection kit (AmershamBiosiences, Piscataway, N.J.).

[0369] To determine the Pro115 cell surface expression level,stimulated and unstimulated LNCaP cells from one well of a 6 wellplate were washed with ice-cold PBS with 1 mM CaCl.sub.2 and 0.5 mMMgCl.sub.2 (PBS/MgCa) and incubated in 1 mL ice-coldSulfo-SS-Biotin (0.5 mg/mL in PBS/MgCa; #21331; Pierce) for 30 minat 4.degree. C. on ice. Cells were washed once with ice cold PBS/25mM Tris and twice with ice-cold PBS/MgCa and incubated in 0.2 mLCell Lysis Buffer for 10 min on ice. Seventy-five ug total proteinswere incubated with 100 ul Streptavidin-Agarose (#20349, Pierce)for 2 hours at 4.degree. C. The agarose beads were washed threetimes in Cell Lysis Buffer and retained proteins were eluted fromthe beads by incubation in 1.times. NuPage Sample Buffersupplemented with reducing agent (Invitrogen). The eluted proteinswere separated by gel electrophoresis and analyzed by westernblotting as described above.

[0370] Table 15 summarizes the intensities of the bands at 54 kDa(full length Pro115) and 38 kDa (N-terminal LDL/SRCR domain)observed in the western blot experiment. Band intensity iscategorized as weak (-), intermediate (+/-), strong (+), or notdetected (ND). The size of the band(s) detected is also indicated.The results show that both, total cellular expression and cellsurface expression of Pro115, are upregulated in LNCaP cells uponstimulation with mibolerone. The results further demonstrate thatthe majority of cell surface Pro115 in stimulated LNCaP cells is inits active (cleaved) conformation.

TABLE-US-00027 TABLE 15 Total cellular expression and cell surfaceexpression of Pro115 in stimulated and unstimulated LNCaP cellsUnstimulated LNCaP Stimulated LNCaP Primary Sample Bands Bands MAbloaded on gel detected Intensity detected Intensity A12.1 Wholecell lysate 54 kDa +/- 54 kDa + 38 kDa +/- 38 kDa + A12.1Biotinylated cell 54 kDa - 54 kDa +/- surface protein 38 kDa - 38kDa + eluted from streptavidin agarose

Example 4

Immunohistochemical Staining of Human Tissues

[0371] To evaluate the expression pattern of Pro115 and the utilityof Pro115 mAbs as staining and imaging agents several human tissueswere evaluated. OCT embedded blocks of normal organ tissues wereobtained from Zoion (Hawthorne, N.Y.). Sections were cut at-20.degree. C. in a cryochamber at a thickness of 5-8 um, andair-dried for 30 minutes at room temperature. Immunohistochemical(IHC) staining was performed using the Immunovision Powervision Kit(Vision Biosystems Inc., Norwell, Mass.). Briefly, slides wererinsed in Tris-Buffered Saline with 0.5% Tween -20 (TBS-T) toremove OCT medium and incubated with a series of primary mAbs for30 minutes at room temperature. Primary antibodies included 10anti-Prof 15 mAbs (10 ug/ml), as well as mouse IgG (10 ug/ml) andanti-E-cadherin (BD Biosciences, 1:500 concentration) as negativeand positive controls, respectively. The slides were thenpost-fixed in 4% paraformaldehyde fixative for 10 minutes at roomtemperature, and rinsed with TBS-T. Endogenous peroxidase activitywas quenched by treating the slides with 3% hydrogen peroxidesolution for 10 minutes. After washing in TBS-T, slides wereincubated with anti-mouse IgG-horseradish peroxidase (HRP)secondary antibody (Vision Biosystems) for 30 minutes at roomtemperature in the dark. The slides were then washed again inTBS-T, and the sections were treated with 3,3'-diaminobenzidinechromagen for 2.about.5 minutes (Vision Biosystems). Finally, theslides were counterstained with hematoxylin before mounting withSupermount (Biogenex, San Ramon, Calif.). Staining was thenobserved using a Zeiss Axioskop 2 microscope, and relative stainingintensity was scored. A summary of the staining is in Table 16below.

TABLE-US-00028 TABLE 16 Pro115 expression in various human tissuesmAb Heart Liver Kidney Stomach Bladder Testis Colon Ovary ProstatePancreas Lung B3.1 - ++ tubing - + + + - +++ ++ ++ 2 + C B7.1 - -focal - - - + - +++ + +/- tubing 1 + C B20.1 - - tubing + - - - -+++ + - 1 + C B23.1 - - - - - - + - +++ - - B29.1 - - tubing epi 1+ C + + + - +++ + +/- 1 + C B31.1 - - - - - - + - +++ + +/- B34.1 -- focal - - +/- + - +++ - - tubing 1 + C B53.1 - - - - - - +/- -+++ + - F2.1 - - +/- - - - - - +++ - +/- F3.11.1 - - +/- - - +/- ++- +++ + + E-cadherin - +++ +++ +++ +++ - +++ - +++ +++ +++ IgG1 - -- - - - - - - - -

[0372] As shown in Table 16, all Pro115 MAbs demonstrated theability to detect Pro115 in human tissues and are useful andstaining or imaging agents. Pro115 staining was strongest inprostate tissue. With the exception of B3.1 all Pro115 MAbsexhibited minimal reactivity with the remainder of the organs inthe panel, demonstrating the specificity of the mAbs to Pro115 andthe restricted expression pattern of Pro115.

Example 5

Killing of Pro115 Expressing Cells

Generation of Stable Pro115-Overexpressing Cells

[0373] DU145 cells (ATCC, Manassas, Va.) were transfected withpLPCX retroviral vectors encoding either protease active fulllength Pro115 (Pro115-WT), protease inactive full length Pro115(Pro115-SA, containing a mutation at amino acid residue 441 fromserine to alanine), or a negative control plasmid encoding alkalinephosphatase (AP). Briefly, Phoenix-Ampho cells (Orbigen, San Diego,Calif.) were transfected with the appropriate vectors usingLipofectamine 2000 transfection reagent (Invitrogen, Carlsbad,Calif.), and incubated at 37.degree. C. in 5% CO2 for 48 hours.Supernatants were then collected from the Phoenix-Ampho cells,filtered through 0.45 uM polysulfonic filters, and diluted 1:1 inDMEM supplemented with 10% fetal bovine serum. Polybrene(Hexadimethrine Bromide; Sigma) was then added to the viralsupernatants at a final concentration of 8 ug/ml. The diluted viralsupernatants were then added to adherent DU145 cells and incubatedat 37.degree. C., 5% CO.sub.2 for 5 hours. Fresh medium was added,and the cells were incubated overnight. The medium was thencompletely aspirated and replaced with fresh DMEM+10% fetal bovineserum and the cells were incubated at 37.degree. C., 5% CO.sub.2for 24 hours. Selection was initiated by adding puromycin(Clontech) to the culture medium at a final concentration of 0.6ug/ml. Following selection and scale-up of the transfected cells,Pro115 overexpression was confirmed by Western immunoblot, livecell immunofluorescence, and FACS analysis.

Killing of Pro115 Expressing Cells

[0374] Experiments were performed by incubating DU145-Pro115 stabletransfectants with Pro115 MAbs premixed with MAb-Zap goatanti-mouse Ig saporin conjugate (Advanced Targeting Systems, SanDiego, Calif.). Cell viability was measured at day 5 to detectkilling effects due to internalization of the Pro115 MAbs.

[0375] On the day before MAb treatment, DU145-AP, DU145-Pro115-SA,and DU145-Pro115-WT cells were seeded on 96 well flat bottomsterile cell culture plates (Corning), in triplicate wells, at 5000cells/100 ul/well in DMEM medium with 10% FBS. Plates wereincubated at 37 .degree. C. in 5% CO.sub.2, overnight. Thefollowing day, 25 ul of Pro115 MAbs diluted in DMEM+10% FBS, withor without MAb-Zap reagent was added to the appropriate wells.Final MAb concentrations were 2.0 .mu.g/mL and 0.4 .mu.g/mL, andthe final concentration of MAb-Zap reagent was 1 .mu.g/mL. Plainmedium with or without MAb-Zap reagent were included as controls.In addition, anti-transferrin receptor (TnfR) and anti-Ricin MAbswere included as positive and negative controls, respectively.Plates were shaken gently for five minutes to mix the reagents andthen incubated at 37.degree. C. in 5% CO.sub.2. After 4 days, cellviability was determined using Cell Titer Glo reagent (Promega).Cell viability was expressed as a percentage of the control wellswith medium alone, as shown in Table 17 below.

TABLE-US-00029 TABLE 17 Growth inhibition of Pro115 expressingcells Percent Growth Inhibition Compared to Wells with Medium AloneDU145-AP DU145-Pro115-SA DU145-Pro115-WT mAb mAb + mAbZap mAb mAb +mAbZap mAb mAb + mAbZap Pro115 alone mAb mAb alone mAb mAb alonemAb mAb mAb (2 ug/ml) (2 ug/ml) (0.4 ug/ml) (2 ug/ml) (2 ug/ml)(0.4 ug/ml) (2 ug/ml) (2 ug/ml) (0.4 ug/ml) A107.1 -4.5 -5.4 1 -0.55.3 4.5 -3.7 -3.7 2.7 A123.1 -0.8 -1.1 3.6 2.7 7.3 11.3 1 1 0 B3.1-0.4 21.5 10 10.3 38.5 24.5 4.5 18 7.6 B7.1 -2 7.7 1.8 4.6 20.6 232.2 8.5 8.5 B20.1 -1.1 -3.5 0 0.5 12.5 19.4 0.5 0.6 7 B23.1 -1.54.4 6.9 9.2 20.3 21.6 3.4 7.4 5.7 B29.1 5.1 11.3 4.5 6.7 19.4 20.55.5 6.3 8.7 B31.1 3 7 2.7 3.8 17.9 9.9 4.6 6 7.3 B34.1 0 -2.3 2-4.4 9.5 11.9 0 4.5 5 B53.1 2.4 4.1 4.9 2.7 12.2 18.2 -2.6 1.2 6F2.1 3.9 17.6 11.4 10.3 12.6 13.6 0.5 16.2 14.4 F3.11.1 8.6 15.510.4 11.1 42.5 48 -1.2 26.7 16.8 Ricin 1.5 6 4.5 8.1 7.1 6.3 2 1.50 TnfR 2.7 73.2 81.4 8 74.1 81.8 3.5 67.7 76.2

[0376] Anti-transferrin receptor MAb resulted in significant cellkilling across all three cell types while anti-Ricin MAb had aminimal effect on cell viability, as expected.

[0377] Pro115 mAbs B7.1, B20.1, B23.1, B29.1, B31.1, F2.1 andF3.11.1 exhibited significant cell killing in DU145-Pro115-SA and-Pro115-WT cells, but had a minimal killing effect on the DU145-APcells, indicating Pro115-specific targeting. In general, cellkilling was greater in the DU145-Pro115-SA cells than in the-Pro115-WT cells, which is likely due to higher expression levelsof the Pro115-SA mutant as determined by FACS stain. Pro115 mAbF3.11.1 exhibited the highest level of cell killing of all thePro115 MAbs, with nearly three times the cell killing effect on thePro115-SA cells compared to the AP cells.

Example 6

Inhibition of Pro115 Protease Activity

Generation of Stable Pro115-Overexpressing Cells

[0378] A cell-based protease assay was utilized to screen Pro115antibodies for inhibition of Pro115 activity. Briefly,DU145-Pro115-WT and DU145-Pro115-SA cells (described above) wereseeded on 96-well black tissue culture plates at a density of50,000 cells per well, and incubated overnight at 37.degree. C. in5% CO.sub.2. The cells were then washed three times with serum-freeDMEM, and Pro115 MAbs diluted in serum-free DMEM were added atfinal concentrations of 50, 10, and 2 ug/ml, in triplicates.Anti-Ricin MAb was added as a negative control MAb, and SerineProtease Inhibitor Cocktail #1 (EMD Biosciences, San Diego, Calif.)at final concentrations of 1.times., 0.2.times., and 0.04.times.was added as positive controls. The cells were incubated with theantibodies for 10 minutes at 37.degree. C. Substrate Boc-QAR-AMC(Bachem) diluted in serum-free DMEM was then added to each well ata final concentration of 250 uM. The antibody and substratesolutions were also added to a plate without cells to provide ameasurement of background signal. Plates were incubated at37.degree. C., 5% CO.sub.2 in the dark for 2 hours. The plates werethen read on a SpectraMAX Gemini EM spectrophotometer (MolecularDevices, Sunnyvale, Calif.) at 370 nm excitation, 450 nm emission,and 435 nm auto-cutoff.

[0379] Data generated from the Pro115 inactive DU145-Pro115-SAcells were used to determine the level of background proteaseactivity in the assay. In general, the signal of DU145-Pro115-WTcells was approximately three times higher than that of theDU145-Pro115-SA cells, indicating that Pro115 activity can bespecifically measured in the assay.

[0380] Data from the DU145-Pro115-WT wells were analyzed bydetermining the average signal from each set of triplicates. Thebackground signal from the cell-free plates was then subtractedout, and the data was expressed as percent inhibition relative tothe untreated DU145-Pro115-WT signal, as shown in Table 18below.

TABLE-US-00030 TABLE 18 Inhibition of Pro115 protease activity byanti-Pro115 antibodies % Protease Activity Inhibition Relative toDU145-Pro115-WT Untreated Control Pro115 Mab 50 ug/ml 10 ug/ml 2ug/ml B3.1 30.7 17.4 9 B7.1 19.1 9.1 -5.9 B20.1 33 34.7 25.4 B23.124 19.8 8.4 B29.1 35.6 27.4 13.9 B31.1 21.6 11.1 11.2 B34.1 23.529.3 14.9 F2.1 16.7 21.2 17.4 F3.11.1 24.9 28.7 33.6 anti-Ricin 3.3N/D N/D Serine Protease 1X 0.2X 0.04X Inhibitor Cocktail #1 94.782.2 46.9 N/D: not determined

[0381] At 50 ug/ml, the Pro115 MAbs tested exhibited proteaseinhibition ranging from 16% to 35%. In addition, most MAbsexhibited a dose dependent effect, as the protease inhibitiondecreased with decreasing MAb concentration. As expected, thenegative control anti-Ricin MAb had an insignificant effect onprotease activity. The positive control Serine Protease InhibitorCocktail #1 had a profound effect on protease activity, reducingactivity by nearly 95% at the highest concentration. However, thisresult is expected, as the reagent is a broad-spectrum inhibitorthat affects many different serine proteases. For example, at1.times., the serine protease inhibitor cocktail showed a nearly100% inhibition of protease activity in DU145-Pro115-SA cells,which express an inactive form of Pro115. Therefore, it is clearthat the inhibitor has an effect on all background serine proteasesas well.

[0382] Additionally, is noted that while anti-Pro115 mAbs F2.1 andF3.11.1 did not have a clear dose dependent effect on proteaseactivity at the concentrations tested, they did demonstrate highlevels of protease inhibition. A dose dependant response to theseantibodies at lower concentrations indicates that less antibody isneeded to inhibit protease activity and that such potent antibodieshave utility as therapeutic agents.

Example 7

Deposits

Deposit of Cell Lines and DNA

[0383] The following hybridoma cell lines were deposited with theAmerican Type Culture Collection (ATCC) located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A., and accorded accessionnumbers.

TABLE-US-00031 TABLE 19 ATCC deposits ATCC Hybridoma Accession No.Deposit Date Pro115.B7.1 PTA-7604 19 May 2006 Pro115.B34.1 PTA-760519 May 2006

[0384] Anti-Pro115 antibody hybridomas Pro115.B7.1 and Pro115.B34.1were shipped to the ATCC via FedEx Overnight on 18 May 2006. APatent Specialist at the ATCC Patent Depository confirmed receiptof the shipment in good condition via email on 19 May 2006.

[0385] The names of the deposited hybridoma cell lines above may beshortened for convenience of reference. E.g. B7.1 corresponds toPro115.B7.1. These hybridomas correspond to the clones (with theirfull names) listed in Table 19.

[0386] These deposits were made under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure and theRegulations there under (Budapest Treaty). This assures maintenanceof viable cultures for 30 years from the date of deposit. Theorganisms will be made available by ATCC under the terms of theBudapest Treaty, and subject to an agreement between diaDexus, Inc.and ATCC, which assures permanent and unrestricted availability ofthe progeny of the cultures to the public upon issuance of thepertinent U.S. patent or upon laying open to the public of any U.S.or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled theretoaccording to 35 USC .sctn.122 and the Commissioner's rules pursuantthereto (including 3 7 CFR .sctn.1.14 with particular reference to886 OG 638).

[0387] The assignee of the present application has agreed that ifthe cultures on deposit should die or be lost or destroyed whencultivated under suitable conditions, they will be promptlyreplaced on notification with a viable specimen of the sameculture. Availability of the deposited strains are not to beconstrued as a license to practice the invention in contraventionof the rights granted under the authority of any government inaccordance with its patent laws. The making of these deposits is byno means an admission that deposits are required to enable theinvention.

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