WO2007016590A2 - Ovr232v3 antibody compositions and methods of use - Google Patents

Abstract

The invention provides isolated anti-ovarian, colon, prostate, or lung cancer antigen (Ovr232v3) antibodies that bind to Ovr232v3 on a mammalian cell. The invention also encompasses compositions containing an anti-Ovr232v3 antibody and a carrier. These compositions can be provided in an article of manufacture or a kit. Another aspect of the invention is an isolated nucleic acid encoding an anti-Ovr232v3 antibody, as well as an expression vector comprising the isolated nucleic acid. Also provided are cells that produce the anti-Ovr232v3 antibodies. The invention encompasses a method of producing the anti-Ovr232v3 antibodies. Other aspects of the invention are a method of killing an Ovr232v3-expressing cancer cell via contacting the cancer cell with an anti-Ovr232v3 antibody and a method of alleviating or treating an Ovr232v3 -expressing cancer in a mammal via administering a therapeutically effective amount of the anti-Ovr232v3 antibody to the mammal.

Description

Ovr232v3 ANTIBODY COMPOSITIONS AND METHODS OF USE

This patent application claims the benefit of priority from U.S. Provisional Application Serial No. 60/705,502, filed July 29, 2005, teachings of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to anti-Ovr232v3 antibody compositions and methods of killing Ovr232v3-expressing ovarian, colon, prostate, or lung cancers cells.

BACKGROUND OF THE INVENTION

Ovarian Cancer

Cancer of the ovaries is the fourth-most common cause of cancer death in women in the United States, with more than 23,000 new cases and roughly 14,000 deaths predicted for the year 2001. Shridhar, V. et al, Cancer Res. 61(15): 5895-904 (2001); Memarzadeh,.S. Sc Berek, J. S., J. Reprod. Med. 46(7): 621-29 (2001). The American Cancer Society (ACS) estimates that there will be about 25,580 new cases of ovarian cancer hi 2004 and ovarian cancer will cause about 16,090 deaths in the United States. ACS Website: cancer with the extension .org of the world wide web. More women die annually from ovarian cancer than from all other gynecologic malignancies combined. The incidence of ovarian cancer in the US is estimated to 14.2 per 100,000 women per year and 9 women per 100, 000 die every year from ovarian cancer. In 2004, approximately 70- 75% of new diagnoses will be stage III and IV carcinoma with a predicted 5-year survival of -15%. Jemal et al., Annual Report to the Nation on the Status of Cancer, 1975-2001, with a Special Feature Regarding Survival. Cancer 2004; 101: 3-27. The incidence of ovarian cancer is of serious concern worldwide, with an estimated 191,000 new cases predicted annually. Runnebaum, I. B. & Stickeler, E., J. Cancer Res. Clin. Oncol. 127(2): 73-79 (2001). Unfortunately, women with ovarian cancer are typically asymptomatic until the disease has metastasized. Because effective screening for ovarian cancer is not available, roughly 70% of women diagnosed have an advanced stage of the cancer with a five-year survival rate of ~25-30%. Memarzadeh, S. & Berek, J. S., supra; Nunns, D. et al., Obstet. Gynecol. Surv. 55(12): 746-51. Conversely, women diagnosed with early stage ovarian cancer enjoy considerably higher survival rates. Werness, B. A. & Eltabbakh, G. H., Int'l. J. Gynecol. Pathol. 20(1): 48-63 (2001). Although our understanding of the etiology of ovarian cancer is incomplete, the results of extensive research in this area point to a combination of age, genetics, reproductive, and dietary/environmental factors. Age is a key risk factor in the development of ovarian cancer: while the risk for developing ovarian cancer before the age of 30 is slim, the incidence of ovarian cancer rises linearly between ages 30 to 50, increasing at a slower rate thereafter, with the highest incidence being among septagenarian women. Jeanne M. Schilder et al., Hereditary Ovarian Cancer; Clinical Syndromes and Management, in Ovarian Cancer 182 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001).

With respect to genetic factors, a family history of ovarian cancer is the most significant risk factor in the development of the disease, with that risk depending on the number of affected family members, the degree of their relationship to the woman, and which particular first degree relatives are affected by the disease. Id. Mutations in several genes have been associated with ovarian cancer, including BRCAl and BRCA2, both of which play a-key role in the development of breast cancer, as well as hMSH2 and hMLHl, both of which are associated with hereditary non-polyposis colon cancer. Katherine Y. Look, Epidemiology, Etiology, and Screening of Ovarian Cancer, in Ovarian Cancer 169, 171-73 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). BRCAl , located on chromosome 17, and BRC A2, located on chromosome 13, are tumor suppressor genes implicated in DNA repair; mutations in these genes are linked to roughly 10% of ovarian cancers. Id. at 171-72; Schilder et al., supra at 185-86. hMSH2 and hMLHl are associated with DNA mismatch repair, and are located on chromosomes 2 and 3, respectively; it has been reported that roughly 3% of hereditary ovarian carcinomas are due to mutations in these genes. Look, supra at 173; Schilder et al., supra at 184, 188-89.

Reproductive factors have also been associated with an increased or reduced risk of ovarian cancer. Late menopause, nulliparity, and early age at menarche have all been linked with an elevated risk of ovarian cancer. Schilder et al., supra at 182. One theory hypothesizes that these factors increase the number of ovulatory cycles over the course of a woman's life, leading to "incessant ovulation," which is thought to be the primary cause of mutations to the ovarian epithelium. Id.; Laura J. Havrilesky & Andrew Berchuck, Molecular Alterations in Sporadic Ovarian Cancer, in Ovarian Cancer 25 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). The mutations may be explained by the fact that ovulation results in the destruction and repair of that epithelium, necessitating increased cell division, thereby increasing the possibility that an undetected mutation will occur. Id. Support for this theory may be found in the fact pregnancy, lactation, and the use of oral contraceptives, all of which suppress ovulation, confer a protective effect with respect to developing ovarian cancer. Id.

Among dietary/environmental factors, there would appear to be an association between high intake of animal fat or red meat and ovarian cancer, while the antioxidant Vitamin A, which prevents free radical formation and also assists in maintaining normal cellular differentiation, may offer a protective effect. Look, supra at 169. Reports have also associated asbestos and hydrous magnesium trisilicate (talc), the latter of which may be present in diaphragms and sanitary napkins. Id. at 169-70.

Current screening procedures for ovarian cancer, while of some utility, are quite limited in their diagnostic ability, a problem that is particularly acute at early stages of cancer progression when the disease is typically asymptomatic yet is most readily treated. Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 166 (1998); Memarzadeh & Berek, supra; Runnebaum & Stickeler, supra; Werness & Eltabbakh, supra. Commonly used screening tests include biannual rectovaginal pelvic examination, radioimmunoassay to detect the CA- 125 serum tumor marker, and transvaginal ultrasonography. Burdette, supra at 166. Currently, CA- 125 is the only clinically approved serum marker for use in ovarian cancer. CA- 125 is found elevated in the majority of serous cancers, but is elevated in only half of those women with early stage disease. The major clinical application of CA125 is in monitoring treatment success or detection of recurrence in women undergoing treatment for ovarian cancer. Markman M. The Oncologist; 2: 6-9 (1997). The use of CA125 as a screening marker is limited because it is frequently elevated in women with benign diseases such as endometriosis. Hence, there is a critical need for novel serum markers that are more sensitive and specific for the detection of ovarian cancer when used alone, or in combination with CAl 25. Bast RC. Et al., Early Detection of Ovarian Cancer: Promise and Reality in Ovarian Cancer- Cancer Research and Treatment VoI 107 (Stack MS, Fishman, DA, eds., 2001). Pelvic examination has failed to yield adequate numbers of early diagnoses, and the other methods are not sufficiently accurate. Id. One study reported that only 15% of patients who suffered from ovarian cancer were diagnosed with the disease at the time of their pelvic examination. Look, supra at 174. Moreover, the CA-125 test is prone to giving false positives in pre-menopausal women and has been reported to be of low predictive value in post-menopausal women. Id. at 174-75. Although transvaginal ultrasonography is now the preferred procedure for screening for ovarian cancer, it is unable to distinguish reliably between benign and malignant tumors, and also cannot locate primary peritoneal malignancies or ovarian cancer if the ovary size is normal. Schilder et al., supra at 194-95. While genetic testing for mutations of the BRCAl , BRC A2, hMSH2, and hMLHl genes is now available, these tests may be too costly for some patients and may also yield false negative or indeterminate results. Schilder et al., supra at 191-94.

Additionally, current efforts focus on the identification of panels of biomarkers that can be used in combination. Bast RC Jr., J Clin Oncol 2003; 21: 200-205. Currently, other markers being evaluated as potential ovarian serum markers which may serve as members of a multi-marker panel tα. improve, detection of ovarian cancer are HE4; mesothelin; kallikrein 5, 8, 10 and 11; and prostasin. Urban et al. Ovarian cancer screening Hematol Oncol Clin North Am. 2003 Aug;17(4):989-1005; Hellstrom et al. The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma, Cancer Res. 2003 JuI l;63(13):3695-700; Ordonez, Application of mesothelin immunostaining in tumor diagnosis, Am J Surg Pathol. 2003 Nov;27(l 1): 1418-28; Diamandis EP et al., Cancer Research 2002; 62: 295-300; Yousef GM et al., Cancer Research 2003; 63: 3958-3965; Kishi T et al., Cancer Research 2003; 63: 2771-2774; Luo LY et al., Cancer Research 2003; 63: 807-811; Mok SC et al., J Natl Cancer Inst 2001; 93 (19): 1437-1439.

The staging of ovarian cancer, which is accomplished through surgical exploration, is crucial in determining the course of treatment and management of the disease. AJCC Cancer Staging Handbook 187 (Irvin D. Fleming et al. eds., 5th ed. 1998); Burdette, supra at 170; Memarzadeh & Berek, supra; Shridhar et al., supra. Staging is performed by reference to the classification system developed by the International Federation of Gynecology and Obstetrics. David H. Moore, Primary Surgical Management of Early Epithelial Ovarian Carcinoma, in Ovarian Cancer 203 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001); Fleming et al. eds., supra at 188. Stage I ovarian cancer is characterized by tumor growth that is limited to the ovaries and is comprised of three substages. Id. In substage IA, tumor growth is limited to one ovary, there is no tumor on the external surface of the ovary, the ovarian capsule is intact, and no malignant cells are present in ascites or peritoneal washings. Id. Substage IB is identical to Al, except that tumor growth is limited to both ovaries. Id. Substage IC refers to the presence of tumor growth limited to one or both ovaries, and also includes one or more of the following characteristics: capsule rupture, tumor growth on the surface of one or both ovaries, and malignant cells present in ascites or peritoneal washings. Id. Stage II ovarian cancer refers to tumor growth involving one or both ovaries, along with pelvic extension. Id. Substage HA involves extension and/or implants on the uterus and/or fallopian tubes, with no malignant cells in the ascites or peritoneal washings, while substage HB involves extension into other pelvic organs and tissues, again with no malignant cells in the ascites or peritoneal washings. Id. Substage HC involves pelvic extension as in HA or HB, but with malignant cells in the ascites or peritoneal washings. Id.

Stage III ovarian cancer involves tumor growtLin one or both ovaries, with peritoneal metastasis beyond the pelvis confirmed by microscope and/or metastasis in the regional lymph nodes. Id. Substage IIIA is characterized by microscopic peritoneal metastasis outside the pelvis, with substage IUB involving macroscopic peritoneal metastasis outside the pelvis 2 cm or less in greatest dimension. Id. Substage HIC is identical to IIIB, except that the metastasis is greater than 2 cm in greatest dimension and may include regional lymph node metastasis. Id. Lastly, Stage IV refers to the presence distant metastasis, excluding peritoneal metastasis. Id. While surgical staging is currently the benchmark for assessing the management and treatment of ovarian cancer, it suffers from considerable drawbacks, including the invasiveness of the procedure, the potential for complications, as well as the potential for inaccuracy. Moore, supra at 206-208, 213. In view of these limitations, attention has turned to developing alternative staging methodologies through understanding differential gene expression in various stages of ovarian cancer and by obtaining various biomarkers to help better assess the progression of the disease. Vartiainen, J. et al., Int'lJ. Cancer, 95(5): 313-16 (2001); Shridhar et al. supra; Baekelandt, M. et al, J Clin. Oncol. 18(22): 3775-81.

The treatment of ovarian cancer typically involves a multiprong attack, with surgical intervention serving as the foundation of treatment. Dennis S. Chi & William J. Hoskins, Primary Surgical Management of Advanced Epithelial Ovarian Cancer, Jn Ovarian Cancer 241 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). For example, in the case of epithelial ovarian cancer, which accounts for ~90% of cases of ovarian cancer, treatment typically consists of: (1) cytoreductive surgery, including total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and lymphadeiiectomy, followed by (2) adjuvant chemotherapy with paclitaxel and either cisplatin or carboplatin. Eltabbakh, G.H. & Awtrey, C. S., Expert Op. Pharmacother. 2(10): 109-24. Despite a clinical response rate of 80% to the adjuvant therapy, most patients experience tumor recurrence within three years of treatment. Id. Certain patients may undergo a second cytoreductive surgery and/or second-line chemotherapy. Memarzadeh & Berek, supra.

From the foregoing, it is clear that procedures used for detecting, diagnosing, monitoring, staging, prognosticating, and preventing the recurrence of ovarian cancer are of critical importance to the outcome of the patient. Moreover, current procedures, while helpful in each of these analyses, are limited by their specificity, sensitivity, invasiveness, and/or their cost. As such, highly specific and sensitive procedures that would operate by way of detecting novel markers in cells, tissues, or bodily fluids, with minimal invasiveness and at a reasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accurate methods for predicting whether a person is likely to develop ovarian cancer, for diagnosing ovarian cancer, for monitoring the progression of the disease, for staging the ovarian cancer, for determining whether the ovarian cancer has metastasized, and for imaging the ovarian cancer. There is also a need for better treatment of ovarian cancer.

Colon Cancer

Colorectal cancer is the second most common cause of cancer death in the United States and the third most prevalent cancer in both men and women. M. L. Davila & A. D. Davila, Screening for Colon and Rectal Cancer, in Colon and Rectal Cancer 47 (Peter S. Edelstein ed., 2000). Colorectal cancer is categorized as a digestive system cancer by the American Cancer Society (ACS) which also includes cancers of the esophagus, stomach, small intestine, anus, anal canal, anorectum, liver and intrahepatic bile duct, gallbladder and other biliary, pancreas, and other digestive organs. The ACS estimates that there will be about 253,500 new cases of digestive system cancers in 2005 in the United States alone. Digestive system cancers will cause an estimated 136,060 deaths combined in the United States in 2005. Specifically, The ACS estimates that there will be about 104,950 new cases of colon cancer, 40,340 new cases of rectal cancer and 5,420 new cases of small intestine cancer in the 2005 in the United States alone. Colon, rectal and small intestine cancers will cause an estimated 57,360 deaths combined in the United States in 2005. ACS Website: cancer with the extension .org of the world wide web. Nearly all cases of colorectal cancer arise from adenomatous polyps, some of which mature into large polyps, undergo abnormal growth and development, and ultimately progress into cancer. Davila at 55-56. This progression would appear to take at least 10 years in most patients, rendering it a readily treatable form of cancer if diagnosed early, when the cancer is localized. Davila at 56; Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment

125 (1998). . . Although, our understanding of the etiology of colon cancer is undergoing continual refinement, extensive research in this area points to a combination of factors, including age, hereditary and nonhereditary conditions, and environmental/dietary factors. Age is a key risk factor in the development of colorectal cancer, Davila at 48, with men and women over 40 years of age become increasingly susceptible to that cancer, Burdette at 126. Incidence rates increase considerably in each subsequent decade of life. Davila at 48. A number of hereditary and nonhereditary conditions have also been linked to a heightened risk of developing colorectal cancer, including familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (Lynch syndrome or HNPCC), a personal and/or family history of colorectal cancer or adenomatous polyps, inflammatory bowel disease, diabetes mellitus, and obesity. Id. at 47; Henry T. Lynch & Jane F. Lynch, Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndromes), in Colon and Rectal Cancer 67-68 (Peter S. Edelstein ed., 2000). Environmental/dietary factors associated with an increased risk of colorectal cancer include a high fat diet, intake of high dietary red meat, and sedentary lifestyle. Davila at 47; Reddy, B. S., Prev. Med. 16(4): 460-7 (1987). Conversely, environmental/dietary factors associated with a reduced risk of colorectal cancer include a diet high in fiber, folic acid, calcium, and hormone-replacement therapy in postmenopausal women. Davila at 50-55. The effect of antioxidants in reducing the risk of colon cancer is unclear. Davila at 53. Because colon cancer is highly treatable when detected at an early, localized stage, screening should be a part of routine care for all adults starting at age 50, especially those with first-degree relatives with colorectal cancer. One major advantage of colorectal cancer screening over its counterparts in other types of cancer is its ability to not only detect precancerous lesions, but to remove them as well. Davila at 56. The key colorectal cancer screening tests in use today are fecal occult blood test, sigmoidoscopy, colonoscopy, double-contrast barium enema, and the carcinoembryonic antigen (CEA) test. Burdette at 125; Davila at 56.

The fecal occult blood test (FOBT) screens for colorectal cancer by detecting the amount of blood in the stool, the premise being that neoplastic tissue, particularly malignant tissue, bleeds more than typical mucosa, with the amount of bleeding increasing with polyp size and cancer stage. Davila at 56-57. While effective at detecting early stage tumors, FOBT is unable to detect adenomatous polyps (premalignant lesions), and, depending on the contents of the fecal sample, is subject to rendering false positives. Davila at 56-59. Sigmoidoscopy and colonoscopy, by contrast, allow direct visualization of the bowel, and enable one to detect, biopsy, and remove adenomatous polyps. Davila at 59-60, 61. Despite the advantages of these procedures, there are accompanying downsides: sigmoidoscopy, by definition, is limited to the sigmoid colon and below, colonoscopy is a relatively expensive procedure, and both share the risk of possible bowel perforation and hemorrhaging. Davila at 59-60. Double-contrast barium enema (DCBE) enables detection of lesions better than FOBT, and almost as well a colonoscopy, but it may be limited in evaluating the winding rectosigmoid region. Davila at 60. The CEA blood test, which involves screening the blood for carcinoembryonic antigen, shares the downside of FOBT, in that it is of limited utility in detecting colorectal cancer at an early stage. Burdette at 125. Once colon cancer has been diagnosed, treatment decisions are typically made in reference to the stage of cancer progression. A number of techniques are employed to stage the cancer (some of which are also used to screen for colon cancer), including pathologic examination of resected colon, sigmoidoscopy, colonoscopy, and various imaging techniques. AJCC Cancer Staging Handbook 84 (Irvin D. Fleming et al. eds., 5^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 resonance imaging are useful in staging colorectal cancer in its later stages, both have unacceptably low staging accuracy for identifying early stages of the disease, due to the difficulty that both methods have in (1) revealing the depth of bowel wall tumor infiltration and (2) diagnosing malignant adenopathy. Thoeni, R. F., Radiol. Clin. N. Am. 35(2): 457-85 (1997). Rather, techniques such as transrectal ultrasound (TRUS) are preferred in this context, although this technique is inaccurate with respect to detecting small lymph nodes that may contain metastases. David Blumberg & Frank G. Opelka, Neoadjuvant and Adjuvant Therapy for Adenocarcinoma of the Rectum, in Colon and Rectal Cancer 316 (Peter S. Edelstein ed., 2000). Several classification systems have been devised to stage the extent of colorectal cancer, including the Dukes' system and the more detailed International Union against Cancer- American Joint Committee on Cancer TNM staging system, which is considered by many in the field to be a more useful staging system. Burdette at 126-27. The TNM system, which is used for either clinical or pathological staging, is divided into four stages, each of which evaluates the extent of cancer growth with respect to primary tumor (T), regional lymph nodes (N), and distant metastasis (M). Fleming at 84-85. The system focuses on the extent of tumor invasion into the intestinal wall, invasion of adjacent structures, the number of regional lymph nodes that have been affected, and whether distant metastasis has occurred. Fleming at 81. Stage 0 is characterized by in situ carcinoma (Tis), in which the cancer cells are located inside the glandular basement membrane (intraepithelial) or lamina propria (intramucosal). In this stage, the cancer has not spread to the regional lymph nodes (NO), and there is no distant metastasis (MO). In stage I, there is still no spread of the cancer to the regional lymph nodes and no distant metastasis, but the tumor has invaded the submucosa (Tl) or has progressed further to invade the muscularis propria (T2). Stage II also involves no spread of the cancer to the regional lymph nodes and no distant metastasis, but the tumor has invaded the subserosa, 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 is characterized by any of the T substages, no distant metastasis, and either metastasis in 1 to 3 regional lymph nodes (Nl) or metastasis in four or more regional lymph nodes (N2). Lastly, stage IV involves any of the T or N substages, as well as distant metastasis. Fleming at 84-85; Burdette at 127.

Currently, pathological staging of colon cancer is preferable over clinical staging as pathological staging provides a more accurate prognosis. Pathological staging typically involves examination of the resected colon section, along with surgical examination of the abdominal cavity. Fleming at 84. Clinical staging would be a preferred method of staging were it at least as accurate as pathological staging, as it does not depend on the invasive procedures of its counterpart.

Turning to the treatment of colorectal cancer, surgical resection results in a cure for roughly 50% of patients. Irradiation is used both preoperatively and postoperatively in treating colorectal cancer. Chemotherapeutic agents, particularly 5-fluorouracil, are also powerful weapons in treating colorectal cancer. Other agents include irinotecan and floxuridine, cisplatin, levamisole, methotrexate, interferon-α, and leucovorin. Burdette at 125, 132-33. Nonetheless, thirty to forty percent of patients will develop a recurrence of colon cancer following surgical resection, which in many patients is the ultimate cause of death. 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 to determine response to therapy and to detect persistent or recurrent disease and metastasis.

The next few paragraphs describe the some of molecular bases of colon cancer. In the case of FAP, the tumor suppressor gene APC (adenomatous polyposis coli), chromosomally located at 5q21, has been either inactivated or deleted by mutation.

Alberts et al, Molecular Biology of the Cell 1288 (3d ed. 1994). The APC protein plays 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 and Rectal Cancer 8 (Peter S. Edelstein, ed., 2000). Of those patients with colorectal cancer who have normal APC genes, over 65% have such mutations in the cancer cells but not in other tissues. Alberts et al., supra at 1288. In the case of HPNCC, patients manifest abnormalities in the tumor suppressor gene HNPCC, but only about 15% of tumors contain the mutated gene. Id. A host of other genes have also been implicated in colorectal cancer, including the K-ras, N-røs, H-ras and c-myc oncogenes, and the tumor suppressor genes DCC (deleted in colon carcinoma) and p5 ^'3. Hall & Madoff, supra at 8-9; Alberts et al., supra at 1288. Abnormalities in Wg/Wnt signal transduction pathway are also associated with the development of colorectal carcinoma. Taipale, J. and Beachy, P.A. Nature 411 : 349-354 (2001). Wntl is a secreted protein gene originally identified within mouse mammary cancers by its insertion into the mouse mammary tumor virus (MMTV) gene. The protein is homologous to the wingless (Wg) gene product of Drosophila, in which it functions as an important factor for the determination of dorsal-ventral segmentation and regulates the formation of fly imaginal discs. Wg/Wnt pathway controls cell proliferation, death and differentiation. Taipal (2001). There are at least 13 members in the Wnt family. These proteins have been found expressed mainly in the central nervous system (CNS) of vertebrates as well as other tissues such as mammary and intestine. The Wnt proteins are the ligands for a family of seven transmembrane domain receptors related to the Frizzled gene product in Drosophila. Binding Wnt to Frizzled stimulates the activity of the downstream target. Dishevelled, which in turn inactivates the glycogen synthesase kinase 3β (GSK3β). Taipal (2001). Usually active GSK3β will form a complex with the adenomatous polyposis coli (APC) protein and phosphorylate another complex member, β-catenin. Once phosphorylated, β-catenin is directed to degradation through the ubiquitin pathway. When GSK3β or APC activity is down regulated, β-catenin is accumulated in the cytoplasm and binds to the T-cell factor or lymphocyte excitation factor (Tcf/Lef) family of transcriptional factors. Binding of β-catenin to Tcf releases the transcriptional repression and induces gene transcription. Among the genes regulated by β-catenin are a transcriptional repressor Engrailed, a transforming growth factor-β (TGF-β) family member Decapentaplegic, and the cytokine Hedgehog in Drosophila. β-Catenin also involves in regulating cell adhesion by binding to α-catenin and E-cadherin. On the other hand, binding of β-catenin to these proteins controls the cytoplasmic β-catenin level and its complexing with TCF. Taipal (2001). Growth factor stimulation and activation of c- src or v-src also regulate β-catenin level by phosphorylation of α-catenin and its related protein, pl20^cas. When phosphorylated, these proteins decrease their binding to E- cadherin and β-catenin resulting in the accumulation of cytoplasmic β-catenin. Reynolds, A.B. et al. MoI. Cell Biol. 14: 8333-8342 (1994). In colon cancer, c-src enzymatic activity has been shown increased to the level of v-src. Alternation of components in the Wg/Wnt pathway promotes colorectal carcinoma development. The best known modifications are to the APC gene. Nicola S et al. Hum. MoI Genet 10:721-733 (2001). This germline mutation causes the appearance of hundreds to thousands of adenomatous polyps in the large bowel. It is the gene defect that accounts for the autosomally dominantly inherited FAP and related syndromes. The molecular alternations that occur in this pathway largely involve deletions of alleles of tumor-suppressor genes, such as APC, p53 and Deleted in Colorectal Cancer (DCC), combined with mutational activation of proto-oncogenes, especially c-Ki-ras. Aoki, T. et al. Human Mutat. 3: 342-346 (1994). All of these lead to genomic instability in colorectal cancers.

Another source of genomic instability in colorectal cancer is the defect of DNA mismatch repair (MMR) genes. Human homologues of the bacterial mutHLS complex (hMSH2, hMLHl, hPMSl, hPMS2 and hMSH6), which is involved in the DNA mismatch repair in bacteria, have been shown to cause the HNPCC (about 70-90% HNPCC) when mutated. Modrich, P. and Lahue, R. Ann Rev. Biochem. 65: 101-133 (1996); and Peltomaki, P. Hum. MoI. Genet 10: 735-740 (2001), The inactivation of these proteins leads to the accumulation of mutations and causes genetic instability that represents errors in the accurate replication of the repetitive mono-, di-, tri- and tetra-nucleotide repeats, which are scattered throughout the genome (microsatellite regions). Jass, J.R. et al. J

Gastroenterol Hepatol 17: 17-26 (2002). Like in the classic FAP, mutational activation of c-Ki-ras is also required for the promotion of MSI in the alternative HNPCC. Mutations in other proteins such as the tumor suppressor protein phosphatase PTEN (Zhou, X.P. et al. Hum. MoI. Genet 11: 445-450 (2002)), BAX (Buttler, L.M. Aus. N. Z. J. Surg. 69: 88- 94 (1999)), Caspase-5 (Planck, M. Cancer Genet Cytogenet. 134: 46-54 (2002)), TGFβ- 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 been found in some colorectal tumors possibly as the cause of MMR defect.

Some tyrosine kinases have been shown up-regulated in colorectal 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 cells may play an important role in the promotion of colorectal cancers through the extracellular matrix (ECM) and integrin-mediated signaling pathways. Jessup, J.M. et al, The molecular biology of colorectal carcinoma, in: The Molecular Basis of Human Cancer, 251-268 (Coleman W.B. and Tsongalis GJ. Eds. 2002). The formation of c-src/FAK complexes may coordinately deregulate VEGF expression and apoptosis inhibition. Recent evidences suggest that a specific signal-transduction pathway for cell survival that implicates integrin engagement leads to FAK activation and thus activates PI-3 kinase and alct. In turn, akt phosphorylates BAD and blocks apoptosis in epithelial cells. The activation of c-src in colon cancer may induce VEGF expression through the hypoxia pathway. Other genes that may be implicated in colorectal cancer include 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-activated receptor-γ (PPAR-γ) (Gelman, L. et al. Cell MoI. Life ScL 55: 932-943 (1999)), IGF-I (Giovannucci (2001)), thymine DNA glycosylase (TDG) (Hardeland, U. et al. Prog. Nucleic Acid Res. MoI. Biol. 68: 235-253 (2001)) and EGF (Mendelsohn, J. Endocrine- Related Cancer 8: 3-9 (2001)).

Gene deletion and mutation are not the only causes for development of colorectal cancers. Epigenetic silencing .by. DNA methylation also accounts for the lost of function of colorectal cancer suppressor genes. A strong association between MSI and CpG island methylation has been well characterized in sporadic colorectal cancers with high MSI but not in those of hereditary origin. In one experiment, DNA methylation of MLHl,

CDKN2A, MGMT, THBSl, RARB, APC, and pl4ARF genes has been shown in 80%, 55%, 23%, 23%, 58%, 35%, and 50% of 40 sporadic colorectal cancers with high MSI 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 and MTHFR are also associated with an increased or decreased colorectal cancer risk. Pistorius, S. et al. Kongressbd Dtsch Ges Chir Kongr 118: 820-824 (2001); and Potter, J.D. J. Natl. Cancer Inst. 91: 916-932 (1999).

From the foregoing, it is clear that procedures used for detecting, diagnosing, monitoring, staging, prognosticating, and preventing the recurrence of colorectal cancer are of critical importance to the outcome of the patient. Moreover, current procedures, while helpful in each of these analyses, are limited by their specificity, sensitivity, invasiveness, and/or their cost. As such, highly specific and sensitive procedures that would operate by way of detecting novel markers in cells, tissues, or bodily fluids, with minimal invasiveness and at a reasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accurate methods for predicting whether a person is likely to develop colorectal cancer, for diagnosing colorectal cancer, for monitoring the progression of the disease, for staging the colorectal cancer, for determining whether the colorectal cancer has metastasized, and for imaging the colorectal cancer. Following accurate diagnosis, there is also a need for less invasive and more effective treatment of colorectal cancer.

Prostate Cancer Prostate cancer is the most prevalent cancer in men and is the second leading cause of death from cancer among males in the United States. AJCC Cancer Staging Handbook 203 (Irvin D. Fleming et al. eds., 5^th ed. 1998); Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 147 (1998). In 1999, it was estimated that 37,000 men in the United States would die as result of prostate cancer. Elizabeth A. Platz et al., & Edward Giovannucci, Epidemiology of and Risk Factors for Prostate Cancer, in Management of Prostate Cancer 21 (Eric A Klein, ed. 2000). More recently, the American Cancer Society estimated there will be 232,090 new cases of prostate cancer and 30,350 deaths in 2005. Additionally, the rate of prostate cancer deaths in the United States for 1997-2001 was 31.5 per 100,000 men, second only to lung and bronchus cancer. American Cancer Society website: cancer with the extension .org of the world wide 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 invasive prostate cancer in his lifetime is one in six. Platz et al., supra at 21.

Although our understanding of the etiology of prostate cancer is incomplete, the results of extensive research in this area point to a combination of age, genetic and environmental/dietary factors. Platz et al., supra at 19; Burdette, supra at 147; Steven K. Clinton, Diet and Nutrition in Prostate Cancer Prevention and Therapy, in Prostate Cancer: a Multidisciplinarv Guide 246-269 (Philip W. Kantoff et al. eds. 1997). Broadly speaking, genetic risk factors predisposing one to prostate cancer include race and a family history of the disease. Platz et al., supra at 19, 28-29, 32-34. Aside from these generalities, a deeper understanding of the genetic basis of prostate cancer has remained elusive. Considerable research has been directed to studying the link between prostate cancer, androgens, and androgen regulation, as androgens play a crucial role in prostate growth and differentiation. Meena Augustus et al., Molecular Genetics and Markers of Progression, in Management of Prostate Cancer 59 (Eric A Klein ed. 2000). While a number of studies have concluded that prostate tumor development is linked to elevated levels of circulating androgen (e.g., testosterone and dihydrotestosterone), the genetic determinants of these levels remain unknown. Platz et al., supra at 29-30.

Several studies have explored a possible link between prostate cancer and the androgen receptor (AR) gene, the gene product of which mediates the molecular and cellular effects of testosterone and dihydrotestosterone in tissues responsive to androgens. Id. at 30. Differences in the number of certain trinucleotide repeats in exon 1, the region involved in transactivational control, have been of particular interest. Augustus et al., supra at 60. For example, these studies have revealed that as the number of CAG repeats decreases the transactivational ability of the gene product increases, as does the risk of prostate cancer. Platz et al., supra at 30-31. Other research has focused on the α- reductase Type 2 gene, the gene which codes for the enzyme that converts testosterone into dihydrotestosterone. Id. at 30. Dihydrotestosterone has greater affinity for the AR than testosterone, resulting in increased transactivation of genes responsive to androgens. Id. While studies have reported differences among the races in the length of a TA dinucleotide repeat in the 3' untranslated region, no link has been established between the length of that repeat and prostate cancer. Id. Interestingly, while ras gene mutations are implicated in numerous other cancers, such mutations appear not to play a significant role in prostate cancer, at least among Caucasian males. Augustus, supra at 52.

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

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

Once the diagnosis of prostate cancer has been made, treatment decisions for the individual are typically linked to the stage of prostate cancer present in that individual, as well as his age and overall health. Burdette, supra at 151. One preferred classification system for staging prostate cancer was developed by the American Urological Association (AUA). Id. at 148. The AUA classification system divides prostate tumors into four broad stages, A to D, which are in turn accompanied by a number of smaller substages. Burdette, supra at 152-153; Anthony V. D'Amico et al., The Staging of Prostate Cancer, in Prostate Cancer: A Multidisciplinary Guide 41 (Philip W. Kantoff et al. eds. 1997). Stage A prostate cancer refers to the presence of microscopic cancer within the prostate gland. D'Amico, supra at 41. This stage is comprised of two substages: Al, which involves less than four well-differentiated cancer foci within the prostate, and A2, which involves greater than three well-differentiated cancer foci or alternatively, moderately to poorly differentiated foci within the prostate. Burdette, supra at 152; D'Amico, supra at 41. Treatment for stage Al preferentially involves following PSA levels and periodic DRE. Burdette, supra at 151. Should PSA levels rise, preferred treatments include radical prostatectomy in patients 70 years of age and younger, external beam radiotherapy for patients between 70 and 80 years of age, and hormone therapy for those over 80 years of age. Id. Stage B prostate cancer is characterized by the presence of a palpable lump within the prostate. Burdette, supra at 152-53; D'Amico, supra at 41. This stage is comprised of three substages: Bl, in which the lump is less than 2 cm and is contained in one lobe of the prostate; B2, in which the lump is greater than 2 cm yet is still contained within one lobe; and B3, in which the lump has spread to both lobes. Burdette, supra, at 152-53. For stages Bl and B2, the treatment again involves radical prostatectomy in patients 70 years of age and younger, external beam radiotherapy for patients between 70 and 80 years of age, and hormone therapy for those over 80 years of age. /<i. at 151. In stage B3, radical prostatectomy is employed if the cancer is well-differentiated and PSA levels are below 15 ng/niL; otherwise, external beam radiation is the chosen treatment option. Id.

Stage C prostate cancer involves a substantial cancer mass accompanied by extraprostatic extension. Burdette, supra at 153; D'Amico, supra at 41. Like stage A prostate cancer, Stage C is comprised of two substages: substage Cl, in which the tumor is relatively minimal, with minor prostatic extension, and substage C2, in which the tumor is large and bulky, with major prostatic extension. Id. The treatment of choice for both substages is external beam radiation. Burdette, supra at 151.

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 is comprised of four substages: (1) DO, in which acid phophatase levels are persistently high, (2) D 1 , in which only the pelvic lymph nodes_^have been, invaded, (3)

D2, in which the lymph nodes above the aortic bifurcation have been invaded, with or without distant metastasis, and (4) D3, in which the metastasis progresses despite intense hormonal therapy. Id. Treatment at this stage may involve hormonal therapy, chemotherapy, and removal of one or both testes. Burdette, supra at 151.

Despite the need for accurate staging of prostate cancer, current staging methodology is limited. The wide variety of biological behavior displayed by neoplasms of the prostate has resulted in considerable difficulty in predicting and assessing the course of prostate cancer. Augustus et al., supra at 47. Indeed, despite the fact that most prostate cancer patients have carcinomas that are of intermediate grade and stage, prognosis for these types of carcinomas is highly variable. Andrew A Renshaw & Christopher L. Corless, Prognostic Features in the Pathology of Prostate Cancer, in Prostate Cancer: A Multidisciplinary Guide 26 (Philip W. Kantoff et al. eds. 1997). Techniques such as transrectal ultrasound, abdominal and pelvic computerized tomography, and MRI have not been particularly useful in predicting local tumor extension. D'Amico, supra at 53 (editors' comment). While the use of serum PSA in combination with the Gleason score is currently the most effective method of staging prostate cancer, id., PSA is of limited predictive value, Augustus et al., supra at 47; Renshaw et al, supra at 26, and the Gleason score is prone to variability and error, King, C. R. & Long, J. P., Int'l J. Cancer 90(6): 326-30 (2000). As such, the current focus of prostate cancer research has been to obtain biomarkers to help better assess the 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).

Accordingly, there is a great need for more sensitive and accurate methods for predicting whether a person is likely to develop prostate cancer, for diagnosing prostate cancer, for monitoring the progression of the disease, for staging the prostate cancer, for determining whether the prostate cancer has metastasized and for imaging the prostate cancer. There is also a need for better treatment of prostate cancer.

Angio genesis in Cancer

Growth and metastasis of solid tumors are also dependent on angiogenesis. 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 to greater than 2 mm must obtain their own blood supply and do so by inducing the growth .of new. capillary blood vessels. Once these new blood vessels become embedded in the tumor, they provide a means for tumor cells to enter the circulation and metastasize to distant sites such as liver, lung or bone. Weidner, N., et al, 1991, The New England Journal of Medicine, 324(1), 1-8.

Angiogenesis, defined as the growth or sprouting of new blood vessels from existing vessels, is a complex process that primarily occurs during embryonic development. The process is distinct from vasculogenesis, in that the new endothelial cells lining the vessel arise from proliferation of existing cells, rather than differentiating from stem cells. The process is invasive and dependent upon proteolysis of the extracellular matrix (ECM), migration of new endothelial cells, and synthesis of new matrix components. Angiogenesis occurs during embryogenic development 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). Under normal physiological conditions in adults, angiogenesis takes place only in very restricted situations such as hair 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 the formation of a migrating column of endothelial cells. Proteolytic activity is focused at the advancing tip of this "vascular sprout", which breaks down the ECM sufficiently to permit the column of cells to infiltrate and migrate. Behind the advancing front, the endothelial cells differentiate and begin to adhere to each other, thus forming a new basement membrane. The cells then cease proliferation and finally define a lumen for the new arteriole or capillary.

Unregulated angiogenesis has gradually been recognized to be responsible for a wide range of disorders, including, but not limited to, cancer, cardiovascular disease, rheumatoid arthritis, psoriasis and diabetic retinopathy. Folkman, 1995, Nat Med 1 (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.

Of particular interest is the observation that angiogenesis is 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 Rev Med 49:407-24. A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay "dormant" without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors Folkman, 1995, supra.

One of the most potent angiogenesis inhibitors is endostatin identified by O'Reilly and Folkman. O'Reilly et al, 1997, Cell 88(2):277-85; O'Reilly et al, 1994, Cell 79(2):3 15-28. Its discovery was based on the phenomenon that certain primary tumors can inhibit the growth of distant metastases. O'Reilly and Folkman hypothesized that a primary tumor initiates angiogenesis by generating angiogenic stimulators in excess of inhibitors. However, angiogenic inhibitors, by virtue of their longer half life in the circulation, reach the site of a secondary tumor in excess of the stimulators. The net result is the growth of primary tumor and inhibition of secondary tumor. Endostatin is one of a growing list of such angiogenesis inhibitors produced by primary tumors. It is a proteolytic fragment of a larger protein: endostatin is a 20 IcDa fragment of collagen XVIII (amino acid Hl 132- K 1315 in murine collagen XVIII). Endostatin has been shown to specifically inhibit endothelial cell proliferation in vitro and block angiogenesis in vivo. More importantly, administration of endostatin to tumor-bearing mice leads to significant tumor regression, and no toxicity or drug resistance has been observed even after multiple treatment cycles. Boehm et al., 1997, Nature 390(6658):404-407. The fact that endostatin targets genetically stable endothelial cells and inhibits a variety of solid tumors makes it a very attractive candidate for anticancer therapy. Fidler and Ellis, 1994, Cell 79(2): 185-8; Gastl et al., 1997, Oncology 54(3):177-84; Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3. In addition, angiogenesis inhibitors have been shown to be more effective when combined with radiation and chemotherapeutic agents. Klement, 2000, J. Clin Invest, 105(8) Rl 5- 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.

As discussed above, each of the methods for diagnosing and staging ovarian, colon, prostate, or lung cancer is limited by the technology employed. Accordingly, there is need for sensitive molecular and cellular markers for the detection of ovarian, colon, prostate, or lung cancer. There is a need for molecular markers for the accurate staging, including clinical and pathological staging, of ovarian, colon, prostate, or lung cancers to optimize treatment methods. In addition, there is a need for sensitive molecular and cellular markers to monitor the progress of cancer treatments, including markers that can detect recurrence of ovarian, colon, prostate, or lung cancers following remission. The present invention provides alternative methods of treating ovarian, colon, prostate, or lung cancer that overcome the limitations of conventional therapeutic methods as well as offer additional advantages that will be apparent from the detailed description below.

SUMMARY OF THE INVENTION This invention is directed to an isolated Ovr232v3 antibody that binds to Ovr232v3 on a mammalian cell. The invention is further directed to an isolated Ovr232v3 antibody that internalizes upon binding to Ovr232v3 on a mammalian cell. The antibody may be a monoclonal antibody. Alternatively, the antibody is an antibody fragment or a chimeric or a humanized antibody. The monoclonal antibody may be produced by a hybridoma selected from the group of hybridomas deposited under American Type Culture Collection on 29 July 2005 comprising Ovr232v3.C31.1 and Ovr232v3.C32.3.

The antibody may compete for binding to the same epitope as the epitope bound by the monoclonal antibody produced by a hybridoma selected from the group of hybridomas deposited under the American Type Culture Collection on 29 July 2005 comprising Ovr232v3.C31.1 and Ovr232v3.C32.3. The invention is also directed to conjugated antibodies. They may be conjugated to a growth inhibitory agent or a cytotoxic agent. The cytotoxic agent may be selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes and toxins. Examples of toxins include, but are not limited to, maytansin, maytansinoids, saporin, gelonin, ricin or calicheamicin. The mammalian cell may be a cancer cell. Preferably, the anti-Ovr232v3 monoclonal antibody that inhibits the growth of O vi^*232 v3 -expressing cancer cells.

The antibody may be produced in bacteria. Alternatively, the antibody may be a humanized form of an anti-Ovr232v3 antibody produced by a hybridoma selected from the group of hybridomas deposited with the ATCC on 29 July 2005 comprising Ovr232v3.C31.1 and Ovr232v3.C32.3.

Preferably, the cancer is selected from the group consisting of ovarian, colon, prostate, and lung cancer. The invention is also directed to a method of producing the antibodies comprising culturing an appropriate cell and recovering the antibody from the cell culture. The invention is also directed to compositions comprising the antibodies and a carrier. The antibody may be conjugated to a cytotoxic agent. The cytotoxic agent may be a radioactive isotope or other chemotherapeutic agent.

The invention is also directed to a method of killing an Ovr232v3 -expressing cancer cell, comprising contacting the cancer cell with the antibodies of this invention, thereby killing the cancer cell. The cancer cell may be selected from the group consisting of ovarian, colon, prostate, and lung cancer cell. The ovarian, colon, prostate or lung may be metastatic cancer. The breast cancer may be HER-2 negative breast cancer. The invention is also directed to a method of alleviating an Ovr232 v3 -expressing cancer in a mammal, comprising administering a therapeutically effective amount of the antibodies to the mammal. In addition, the invention is directed to an article of manufacture comprising a container and a composition contained therein, wherein the composition comprises an antibody as described herein. The article of manufacture may also comprise an additional component, e.g., a package insert indicating that the composition can be used to treat ovarian, colon, prostate, or lung cancer.

BRIEF DESCRIPTION OF THE FIGURES

Figures Ia, Ib, Ic and Id show Ovr232v3 mAbs are internalized by Ovr232v3 expressing cancer cells (HCTl 16).

Figures 2a, 2b, 2c and 2d show Ovr232v3 mAbs are internalized by Ovr232v3 expressing cancer cells (SW620). Figures 3a, 3b, 3c and 3d show Ovr232v3 mAbs are not internalized by cancer cells not expressing Ovr232v3 (HeLa).

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Human "Ovr232v3" as used herein, refers to a protein of 390 amino acids that is expressed on the cell surface as a glycoprotein, whose nucleotide and amino acid sequence sequences are as disclosed in e.g., WO 2004/053079 A2, Cancer specific gene (CSG)

Ovr232v3; and WO 2004/023973 A2, Human diagnostic and therapeutic protein SEQ ID

NO:3891; the disclosures of which are hereby expressly incorporated by reference.

Amino acids 1-341, or 24-341 (without the secretory signal peptide at amino acids 1-23) of Ovr232v3 are expressed on the cell surface. Ovr232v3 as used herein include allelic variants and conservative substitution mutants of the protein which have Ovr232v3 biological activity.

Ovr232v3 is related to the human epithelial cell adhesion molecule (Ep-CAM), also known as Homo sapiens tumor-associated calcium signal transducer 1 (TACSTDl) in the RefSeq database as accessions NM_002354 and NP_002345 (accessible at ncbi with the extension .nlm.nih.gov of the world wide web ). Other synonyms include: EGP, KSA, M4S1, MK-I, EGP40, MICl 8, TROPl, Ep-CAM₅ liEGP-2, CO17-1A, and GA733-2. The refseq database includes the following summary of Ep-CAM:

This 9-exon gene encodes a carcinoma-associated antigen and is a member of a family that includes at least two type I membrane proteins. This antigen is expressed on most normal epithelial cells and gastrointestinal carcinomas and functions as a homotypic calcium-independent cell adhesion molecule. The antigen is being used as a target for immunotherapy treatment of human carcinomas.

Many publications have described the identification, characterization, association with carcinomas, and clinical development of Ep-CAM as a molecular target for cancer therapy and cancer vaccination including the following which are hereby incorporated by reference in their entirety.

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Litvinov SV, van Driel W, van Rhijn CM, Bakker HA, van Krieken H, Fleuren GJ, Warnaar SO. Expression of Ep-CAM in cervical squamous epithelia correlates with an increased proliferation and the disappearance of markers for terminal differentiation. Am J Pathol. 1996 Mar;148(3)865- 75.

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Songun I, van de Velde CJ, Hermans J, Pals ST, Verspaget HW, Vis AN, Menon AG, Litvinov SV, van Krieken JH. Expression of oncoproteins and the amount of eosinophilic and lymphocytic infiltrates can be used as prognostic factors in gastric cancer. Dutch Gastric Cancer Group (DGCG). Br J Cancer. 1996 Dec; 74(11)1783-8.

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Ras E, van der Burg SH, Zegveld ST, Brandt RM, Kuppen PJ, Offringa R, Warnarr SO, van de Velde CJ, Melief CJ. Identification of potential HLA-A *0201 restricted CTL epitopes derived from the epithelial cell adhesion molecule (Ep-CAM) and the carcinoembryonic antigen (CEA). Hum Immunol. 1997 Mar;53(1)81-9.

Smith GK, Banks S, Blumenkopf TA, Cory M, Humphreys J, Laethem RM, Miller J, Moxham CP, Mullin R, Ray PH, Walton LM, Wolfe LA 3rd. Toward antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate. J Biol Chem. 1997 Jun 20;272(25)15804-16.

Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, Prins F, Fleuren GJ, Warnaar SO. Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins. J Cell Biol. 1997 Dec 1 ;139(5)1337-48.

Lujten .RM, Warnaar SO,. Schuurman J, Pasmans SG, Latour S,-Daeron-M, Fleuren GJ_r Litvinov - SV. Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon Rl bearing cells. Hum Antibodies. 1997;8(4)169-80.

Cirulli V, Crisa L, Beattie GM, MaIIy Ml, Lopez AD, Fannon A, Ptasznik A, Inverardi L, Ricordi C, Deerinck T, Ellisman M, Reisfeld RA, Hayek A. KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells morphoregulatory roles in pancreatic islet development. J Cell Biol. 1998 Mar 23; 140(6)1519-34.

Balzar M, Bakker HA, Briaire-de-Bruijn IH, Fleuren GJ, Warnaar SO, Litvinov SV. Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule. MoI Cell Biol. 1998 Aug; 18(8)4833-43.

Velders MP, van Rhijn CM, Oskam E, Fleuren GJ, Warnaar SO, Litvinov SV. The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity relevance for immunotherapy of carcinomas. Br J Cancer. 1998 Aug;78(4)478-83.

Balzar M, Prins FA, Bakker HA, Fleuren GJ, Warnaar SO, Litvinov SV. The structural analysis of adhesions mediated by Ep-CAM. Exp Cell Res. 1999 Jan 10;246(1)108-21.

Takes RP, Baatenburg de Jong RJ, Keuning J, Hermans J, Schuuring E, Van Krieken HJ. Protein expression of cancer associated genes biopsy material compared to resection material in laryngeal cancer. Anticancer Res. 1998 Nov-Dec;18(6B)4787-91.

Takes RP, Baatenburg de Jong RJ, Schuuring E, Litvinov SV, Hermans J, Van Krieken JH. Differences in expression of oncogenes and tumor suppressor genes in different sites of head and neck squamous cell. Anticancer Res. 1998 Nov-Dec;18(6B)4793-800.

HuIs GA, Heijnen IA, Cuomo ME, Koningsberger JC, Wiegman L, Boel E, van derVuurst de Vries AR, Loyson SA, Helfrich W, van Berge Henegouwen GP, van Meijer M, de Kruif J, Logtenberg T. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage- displayed antibody fragments. Nat Biotechnol. 1999 Mar; 17(3)276-81. de Boer CJ, van Krieken JH, Janssen-van Rhijn CM, Litvinov SV. Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J Pathol. 1999 Jun; 188(2)201 -6. Kubuschok B, Passlick B, Izbicki JR, Thetter O, Pantel K. Disseminated tumor cells in lymph nodes as a determinant for survival in surgically resected non-small-cell lung cancer. J Clin Oncol. 1999 Jan; 17(1)19-24. ___^

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McLaughlin PM, Kroesen BJ, Dokter WH, van der Molen H, de Groot M, Brinker MG, Kok K, Ruiters MH, Buys CH, de Leij LF. An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy. Cancer Immunol Immunother. 1999 Sep;48(6)303-11.

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Taguchi N, Hashimoto Y, Naiki M, Farr AG, Boyd RL, Ansari AA, Shultz LD, Kotzin BL, Dorshkind K, lkehara S, Gershwin ME. Abnormal thymic expression of epithelial cell adhesion molecule (EP- CAM) in New Zealand Black (NZB) mice. J Autoimmun. 1999 Dec; 13(4)393-404.

Topping KP, Hough VC, Monson JR, Greenman J. Isolation of human colorectal tumour reactive antibodies using phage display technology, lnt J Oncol. 2000 Jan; 16(1)187-95.

Balzar M, Winter MJ, de Boer CJ, Litvinov SV. The biology of the 17-1 A antigen (Ep-CAM). J MoI Med. 1999 Oct;77(10)699-712. Review.

Anderson R, Schaible K, Heasman J, Wylie C. Expression of the homophilic adhesion molecule, Ep-CAM, in the mammalian germ line. J Reprod Fertil. 1999 JuI; 116(2)379-84.

Verdegaal EM, Huinink DB, Hoogstraten C, Marijnissen AK, Gorsira MB, CIaas FH, Osanto S. Isolation of broadly reactive, tumor-specific, HLA Class-I restricted CTL from blood lymphocytes of a breast cancer patient. Hum Immunol. 1999 Dec;60(12)1195-206.

MacDougall JR, Matrisian LM. Targets of extinction identification of genes whose expression is repressed as a consequence of somatic fusion between cells representing basal and luminal mammary epitheliaLphenotypes- J Cell .Sci-2000 Feb; 113 ( Pt 3)409-23.

Corver WE, Koopman LA, van der Aa J, Regensburg M, Fleuren GJ, Cornelisse CJ. Four-color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer. Cytometry. 2000 Feb 1 ;39(2)96-107.

Kraeft SK, Sutherland R, Gravelin L, Hu GH, Ferland LH, Richardson P, Elias A, Chen LB. Detection and analysis of cancer cells in blood and bone marrow using a rare event imaging system. Clin Cancer Res. 2000 Feb;6(2)434-42.

Zhong XY, Kaul S, Eichler A, Bastert G. Evaluating GA733-2 mRNA as a marker for the detection of micrometastatic breast cancer in peripheral blood and bone marrow. Arch Gynecol Obstet. 1999 Nov;263(1-2)2-6.

Tomita Y, Arakawa F, Yamamoto T, Kuwahara M, Watanabe R, Iwasaki H, Kikuchi M, Kuroki Molecular identification of a human carcinoma-associated glycoprotein antigen recognized by mouse monoclonal antibody FU-MK-1. Jpn J Cancer Res. 2000 Feb;91 (2)231-8.

Ruck P, Wichert G, Handgretinger R, Kaiserling E. Ep-CAM in malignant liver tumours. J Pathol. 2000 May;191(1)102-3. No abstract available.

Piyathilake CJ, Frost AR, Weiss H, Manne U, Heimburger DC, Grizzle WE. The expression of Ep- CAM (17-1A) in squamous cell cancers of the lung. Hum Pathol. 2000 Apr;31 (4)482-7.

Grunberg E, Eckert K, Karsten U, Maurer HR. Effects of differentiation inducers on cell phenotypes of cultured nontransformed and immortalized mammary epithelial cells a comparative immunocytochemical analysis. Tumour Biol. 2000 Jul-Aug;21 (4)211-23.

Basak S, Eck S, Gutzmer R, Smith AJ, Birebent B, Purev E, Staib L, Somasundaram R, Zaloudik J, Li W, Jacob L, Mitchell E, Speicher D, Herlyn D. Colorectal cancer vaccines antiidiotypic antibody, recombinant protein, and viral vector. Ann N Y Acad Sci. 2000 Jun;910237-52; discussion 252-3.

Romeu Figuerola C, Nadal Serra A, Farre Pueyo X, Palomar Garcia V. [Study of cell adhesion in cancer of the larynx] Acta Otorrinolaringol Esp. 2000 May;51 (4)288-92. Spanish. Nagorsen D, Keilholz U, Rivoltini L₁ Schmittel A, Letsch A, Asemissen AM, Berger G, Buhr HJ, Thiel E, Scheibenbogen C. Natural T-cell response against MHC class I epitopes of epithelial cell adhesion molecule, her-2/neu, and carcinoembryonic antigen in patients with colorectal cancer. ancer Res. 2000 Sep 1:60(17)4850-4.

Trebak M, Begg GE, Chong JM, Kanazireva EV, Herlyn D, Speicher DW. Oligomeric state of the colon carcinoma-associated glycoprotein GA733-2 (Ep-C AM/EG P40) and its role in GA733- mediated homotypic cell-cell adhesion. J Biol Chem. 2001 Jan 19;276(3)2299-309. Epub 2000 Oct 31.

Chong JM, Speicher DW. Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep- AM). J Biol Chem. 2001 Feb 23;276(8)5804-13. Epub 2000 Nov 15.

Gastl G, Spizzo G, Obrist P, Dunser M, Mikuz G. Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet. 2000 Dec 9;356(9246)1981-2.

Todorovska A, Roovers RC, Dolezal O, Kortt AA, Hoogenboom HR, Hudson PJ. Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J Immunol Methods. 2001 Feb 1:248(1-2)47-66. Review.

Abicht A, Lochmuller H. Technology evaluation edrecolomab, Centocor Inc. Curr Opin MoI Ther. 2000 Oct;2(5)593-600. Review.

Houba PH, Boven E, van der Meulen-Muileman IH, Leenders RG, Scheeren JW, Pinedo HM, Haisma HJ. Pronounced antitumor efficacy of doxorubicin when given as the prodrug DOX-GA3 in ombination with a monoclonal antibody beta-glucuronidase conjugate, lnt J Cancer. 2001 Feb 15:91(4)550-4.

Balzar M, Briaire-de Bruijn IH, Rees-Bakker HA, Prins FA, Helfrich W, de Leij L, Riethmuller G, Albert! S, Warnaar SO, Fleuren GJ, Litvinov SV. Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions. MoI Cell Biol. 2001 Apr;21 (7)2570-80.

Roovers RC, van der Linden E, de Bruine AP, Arends JW, Hoogenboom HR. Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma. Eur J Cancer. 2001 Mar;37(4)542-9.

Haller DG. Update of clinical trials with edrecolomab a monoclonal antibody therapy for colorectal cancer. Semin Oncol. 2001 Feb;28(1 Suppl 1)25-30.

Staib L, Birebent B, Somasundaram R, Purev E, Braumuller H, Leeser C, Kuttner N, Li W, Zhu D, Diao J, Wunner W, Speicher D, Beger HG, Song H, Herlyn D. lmmunogenicity of recombinant GA733-2E antigen (CO17-1A, EGP, KS1-4, KSA, Ep-CAM) in gastro-intestinal carcinoma patients. lnt J Cancer. 2001 Apr 1 ;92(1)79-87.

Xiang R, Silletti S, Lode HN, Dolman CS, Ruehlmann JM, Niethammer AG, Pertl U, Gillies SD, Primus FJ, Reisfeld RA. Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice. Clin Cancer Res. 2001 Mar;7(3 Suppl)856s-864s.

Calabrese G, Crescenzi C, Morizio E, Palka G, Guerra E, Alberti S. Assignment of TACSTD1 (alias TROP1 , M4S1) to human chromosome 2p21 and refinement of mapping of TACSTD2 (alias TROP2, M1S1) to human chromosome 1 p32 by in situ hybridization. Cytogenet Cell Genet. 2001 ;92(1-2)164-5. No abstract available.

Roovers RC, van der Linden E, de Bruine AP, Arends JW, Hoogenboom HR. In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody. Cancer Immunol Immunother. 2001 Mar;50(1)51-9.

McLaughlin PM, Harmsen MC, Dokter WH, Kroesen BJ, van der Molen H, Brinker MG, Hollema H, Ruiters MH, Buys CH, de Leij LF. The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice a new model to study carcinoma- directed immunotherapy. Cancer Res. 2001 May 15;61 (10)4105-11.

Trojan A, Witzens M, Schultze JL, Vonderheide RH, Harig S, Krackhardt AM, Stahel RA, Gribben JG. Generation of cytotoxic T lymphocytes against native and altered peptides of human leukocyte antigen-A*0201 restricted epitopes from the human epithelial cell adhesion molecule. Cancer Res. 2001 Jun 15;61(12)4761-5.

HuIs G, Gestel D, van der Linden J, Moret E, Logtenberg T. Tumor cell killing by in vitro affinity- matured recombinant human monoclonal antibodies. Cancer Immunol Immunother. 2001 May;50(3)163-71. Meyaard L, van der Vuurst de Vries AR, de Ruiter T, Lanier LL, Phillips JH, Clevers H. The epithelial cellular adhesion molecule (Ep-CAM) is a ligand for the leukocyte-associated immunoglobulin-like receptor (LAIR). J Exp Med. 2001 JuI 2;194(1)107-12. Retraction in Meyaard L, van der Vuurst de Vries AR, de Ruiter T, Lanier LL, Phillips JH, Clevers H. J Exp Med. 2003 Oct 6;198(7)1129.

Takes RP, Baatenburg de Jong RJ, Wijffels K, Schuuring E, Litvinov SV, Hermans J, van Krieken JH. Expression of genetic markers in lymph node metastases compared with their primary tumours in head and neck cancer. J Pathol. 2001 JuI; 194(3)298-302. van der Wee KS, Johnson EW, Dirami G, Dym TM, Hofmann MC. lmmunomagnetic isolation and long-term culture of mouse type A spermatogonia. J Androl. 2001 Jul-Aug;22(4)696-704.

Schwartzberg LS. Clinical experience with edrecolomab a monoclonal antibody therapy for colorectal carcinoma. Crit Rev Oncol Hematol. 2001 Oct;40(1)17-24. Review.

Guillemot JC, Naspetti M, Malergue F, Montcourrier P, Galland F, Naquet P. Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers. Histochem Cell Biol. 2001 Oct; 116(4)371-8.

Gelderman KA, Kuppen PJ, Bruin W, Fleuren GJ, Gorter A. Enhancement of the complement activating capacity of 17-1 A mAb to overcome the effect of membrane-bound complement regulatory proteins on colorectal carcinoma. Eur J Immunol. 2002 Jan;32(1)128-35.

Spizzo G, Obrist P, Ensinger C, Theurl I, Dunser M, Ramoni A, Gunsilius E, Eibl G, Mikuz G, Gast! G. Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer, lnt J Cancer. 2002 Apr 20;98(6)883-8.

Trojan A, Tun-Kyi A, Odermatt B, Nestle FO, Stahel RA. Functional detection of epithelial cell adhesion molecule specific cytotoxic T lymphocytes in patients with lung cancer, colorectal cancer and in healthy donors. Lung Cancer. 2002 May;36(2)151-8.

Mori K, Chano T, Kushima R, Hukuda S, Okabe H. Expression of E-cadherin in chordomas diagnostic marker and possible role of tumor cell affinity. Virchows Arch. 2002 Feb;440(2) 123-7. Erratum in Virchows Arch 2002 Jul;441(1)101.

Lammers R, Giesert C, Grunebach F, Marxer A, Vogel W, Buhring HJ. Monoclonal antibody 9C4 recognizes epithelial cellular adhesion molecule, a cell surface antigen expressed in early steps of erythropoiesis. ExpJHematoL 2002Jun;30(6)537_^45. -

Shawler DL, Bartholomew RM, Garrett MA, Trauger RJ, Dorigo O, Van Beveren C, Marchese A, Ferre F, Duffy C, Carlo DJ, Sherman LA, Gold DP, Sobol RE. Antigenic and immunologic characterization of an allogeneic colon carcinoma vaccine. Clin Exp Immunol. 2002 JuI; 129(1)99- 106.

Naundorf S, Preithner S, Mayer P, Lippold S, WoIf A, Hanakam F, Fichtner I, Kufer P, Raum T, Riethmuller G, Baeuerle PA, Dreier T. In vitro and in vivo activity of MT201 , a fully human monoclonal antibody for pancarcinoma treatment, lnt J Cancer. 2002 Ju1 1;100(1)101-10.

Moore TJ, de Boer-Brouwer M, van Dissel-Emiliani FM. Purified gonocytes from the neonatal rat form foci of proliferating germ cells in vitro. Endocrinology. 2002 Aug;143(8)3171-4.

Giuili G, Tomljenovic A, Labrecque N, Oulad-Abdelghani M, Rassoulzadegan M, Cuzin F. Murine spermatogonia! stem cells targeted transgene expression and purification in an active state. EMBO Rep. 2002 Aug;3(8)753-9. Epub 2002 JuI 15.

Abe H, Kuroki M, lmakiire T, Yamauchi Y, Yamada H, Arakawa F, Kuroki M. Preparation of recombinant MK-1/Ep-CAM and establishment of an ELISA system for determining soluble MK- 1/Ep-CAM levels in sera of cancer patients. J Immunol Methods. 2002 Dec 15;270(2)227-33.

Schweizer C, Strauss G, Lindner M, Marme A, Deo YM, Moldenhauer G. Efficient carcinoma cell killing by activated polymorphonuclear neutrophils targeted with an Ep-CAMxCD64 (HEA125x197) bispecific antibody. Cancer Immunol Immunother. 2002 Dec;51(11-12)621-9. Epub 2002 Oct 8.

Spizzo G, Gastl G, Wolf D, Gunsilius E, Steurer M, Fong D, Amberger A, Margreiter R, Obrist P. Correlation of COX-2 and Ep-CAM overexpression in human invasive breast cancer and its impact on survival. Br J Cancer. 2003 Feb 24;88(4)574-8.

Nasr AF, Nutini M, Palombo B, Guerra E, Albert! S. Mutations of TP53 induce loss of DNA methylation and amplification of the TROP1 gene. Oncogene. 2003 Mar 20;22(11)1668-77.

Ammons WS, Bauer RJ, Horwitz AH, Chen ZJ, Bautista E, Ruan HH, Abramova M, Scott KR, Dedrick RL. In vitro and in vivo pharmacology and pharmacokinetics of a human engineered monoclonal antibody to epithelial cell adhesion molecule. Neoplasia. 2003 Mar-Apr; 5(2) 146-54. Nagorsen D, Scheibenbogen C, Schaller G₁ Leigh B, Schmittel A₁ Letsch A, Thiel E, Keilholz U. Differences in T-cell immunity toward tumor-associated antigens in colorectal cancer and breast iancer patients, lnt J Cancer. 2003 Jun 10; 105(2)221 -5.

Wimberger P, Xiang W, Mayr D, Diebold J, Dreier T, Baeuerle PA, Kimmig R. Efficient tumor cell lysis by autologous, tumor-resident T lymphocytes in primary ovarian cancer samples by an EP- :AM-/CD3-bispecific antibody, lnt J Cancer. 2003 Jun 10;105(2)241-8.

Winter MJ, Nagelkerken B, Mertens AE, Rees-Bakker HA, Briaire-de Bruijn IH, Litvinov SV. Expression of Ep-CAM shifts the state of cadherin-mediated adhesions from strong to weak. Exp ell Res. 2003 Apr 15;285(1)50-8.

Thurmond LM, Stimmel JB, Ingram AC, Ryan CH, Murray DM, Eberwein DJ, Witherspoon SM, Knick VC. Adenocarcinoma cells exposed in vitro to Navelbine or Taxol increase Ep-CAM expression through a novel mechanism. Cancer Immunol Immunother. 2003 Jul;52(7)429-37. Epub 2003 Apr 15.

Roggel F, Hocke S, Lindemann K, Sinz S, WeIk A, Bosl M, Pabst M, Nusser N, Braun S, Schmitt M, Harbeck N. Minimal residual disease in breast cancer and gynecological malignancies phenotype and clinical relevance. Recent Results Cancer Res. 2003;16289-100. Review.

Xiang W, Wimberger P, Dreier T, Diebold J, Mayr D, Baeuerle PA, Kimmig R. Cytotoxic activity of novel human monoclonal antibody MT201 against primary ovarian tumor cells. J Cancer Res Clin Oncol. 2003 Jun;129(6)341-8. Epub 2003 Jun 18.

Kurzen H, Kaul S, Egner U, Deichmann M, Hartschuh W. Expression of MUC 1 and Ep-CAM in Merkel cell carcinomas implications for immunotherapy. Arch Dermatol Res. 2003 Aug;295(4)146- 54. Epub 2003 JuI 3,

Ullenhag GJ, Frodin JE, Mosolits S, Kiaii S, Hassan M, Bonnet MC, Moingeon P, Mellstedt H, Rabbani H. Immunization of colorectal carcinoma patients with a recombinant canarypox virus expressing the tumor antigen Ep-CAM/KSA (ALVAC-KSA) and granulocyte macrophage colony- stimulating factor induced a tumor-specific cellular immune response. Clin Cancer Res. 2003 Jul;9(7)2447-56.

Thurm H, Ebel S, Kentenich C, Hemsen A, Riethdorf S, Coith C, Wallwiener D, Braun S, Oberhoff C, Janicke F, Pantel K. Rare expression of epithelial cell adhesion molecule on residual mjcrometastatic breast cancer ceils .after adjuv.antchemotherapy. Clin CancerRes. 2003 Jul;9(7)2598-604.

Di Paolo C, Willuda J, Kubetzko S, Lauffer I, Tschudi D, Waibel R, Pluckthun A, Stahel RA, Zangemeister-Wittke U. A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity. Clin Cancer Res. 2003 Jul;9(7)2837-48. Erratum in Clin Cancer Res. 2004 Apr 1 ; 10(7)2579.

Ricciardolo FL, Di Stefano A, van Krieken JH, Sont JK, van Schadewijk A, Rabe KF, Donner CF, Hiemstra PS, Sterk PJ, Mauad T. Proliferation and inflammation in bronchial epithelium after allergen in atopic asthmatics. Clin Exp Allergy. 2003 Jul;33(7)905-11.

Yang JZ, Zhang XH, Wu WX, Yan X, Liu YL, Wang JL, Wang FR. [Expression of EP-CAM, beta- catenin in the carcinogenesis of squamous cell carcinoma of uterine cervix] Zhonghua Zhong Liu Za Zhi. 2003 Jul;25(4)372-5. Chinese.

Gires O, Eskofier S, Lang S, Zeidler R, Munz M. Cloning and characterisation of a 1.1 kb fragment of the carcinoma-associated epithelial cell adhesion molecule promoter. Anticancer Res. 2003 Jul-Aug;23(4)3255-61.

Qu N, de Haan A, Harmsen MC, Kroese FG, de Leij LF, Prop J. Specific immune responses against airway epithelial cells in a transgenic mouse-trachea transplantation model for obliterative airway disease. Transplantation. 2003 Oct 15;76(7)1022-8.

Kim JH, Herlyn D, Wong KK, Park DC, Schorge JO, Lu KH, Skates SJ, Cramer DW, Berkowitz RS, Mok SC. Identification of epithelial cell adhesion molecule autoantibody in patients with ovarian cancer. Clin Cancer Res. 2003 Oct 15;9(13)4782-91.

Winter MJ, Nagtegaal ID, van Krieken JH, Litvinov SV. The epithelial cell adhesion molecule (Ep- CAM) as a morphoregulatory molecule is a tool in surgical pathology. Am J Pathol. 2003 Dec; 163(6)2139-48. Review.

Garcia-Caballero T, Pintos E, Gallego R, Parrado C, Blanco M, Bjornhagen V, Forteza J, Beiras A. MOC-31/Ep-CAM immunoreactivity in Merkel cells and Merkel cell carcinomas. Histopathology. 2003 Nov;43(5)480-4. Kirman I₁ Jenkins D, Fowler R₁ Whelan RL. Naturally occurring antibodies to epithelial cell adhesion molecule (EpCAM). Dig Pis ScL 2003 Dec;48(12)2306-9. •

Neidhart J, Allen KO, Barlow DL, Carpenter M, Shaw DR, Triozzi PL, Conry RM. Immunization of colorectal cancer patients with recombinant baculovirus-derived KSA (Ep-CAM) formulated with monophosphoryl lipid A in liposomal emulsion, with and without granulocyte-macrophage colony- stimulating factor. Vaccine. 2004 Jan 26;22(5-6)773-80.

Ruan HH, Scott KR, Bautista E, Ammons WS. ING-i(heMAb), a monoclonal antibody to epithelial cell adhesion molecule, inhibits tumor metastases in a murine cancer model. Neoplasia. 2003 Nov-Dec;5(6)489-94.

Seligson DB, Pantuck AJ, Liu X, Huang Y, Horvath S, Bui MH, Han KR, Correa AJ, Eeva M, Tze S, Belldegrun AS, Figlin RA. Epithelial cell adhesion molecule (KSA) expression pathobiology and its role as an independent predictor of survival in renal cell carcinoma. Clin Cancer Res. 2004 Apr 15;10(8)2659-69.

Varga M, Obrist P, Schneeberger S, Muhlmann G, Felgel-Farnholz C, Fong D, Zitt M, Brunhuber T, Schafer G, Gastl G, Spizzo G. Overexpression of epithelial cell adhesion molecule antigen in gallbladder carcinoma is an independent marker for poor survival. Clin Cancer Res. 2004 May 1 ;10(9)3131-6.

Heinzelmann-Schwarz VA, Gardiner-Garden M, Henshall SM, Scurry J, Scolyer RA, Davies MJ, Heinzelmann M, Kalish LH, Bali A, Kench JG, Edwards LS, Vanden Bergh PM, Hacker NF, Sutherland RL, O'Brien PM. Overexpression of the cell adhesion molecules DDR1 , Claudin 3, and Ep-CAM in metaplastic ovarian epithelium and ovarian cancer. Clin Cancer Res. 2004 JuI 1;10(13)4427-36.

McLaughlin PM, Trzpis M, Kroesen BJ, Helfrich W, Terpstra P, Dokter WH, Ruiters MH, de Leij LF, Harmsen MC. Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines. Cancer Gene Ther. 2004 Sep;11 (9)603-12.

Mosolits S, Markovic K, Frodin JE, Virving L, Magnusson CG, Steinitz M, Fagerberg J, Mellstedt H. Vaccination with Ep-CAM protein or anti-idiotypic antibody induces Th1-biased response against MHC class I- and ll-restricted Ep-CAM epitopes in colorectal carcinoma patients. Clin Cancer Res. 2004 Aug 15; 10(16)5391 -402.

Ryu BY, Orwig KE. Kubota.H, AvarboclcMR,.Brinster.RL. Phenotypic and functional characteristics of spermatogonia! stem cells in rats. Dev Biol. 2004 Oct 1 ;274(1)158-70.

Mosolits S, Campbell F, Litvinov SV, Fagerberg J, Crowe JS, Meilstedt H, Ellis JH. Targeting human Ep-CAM in transgenic mice by anti-idiotype and antigen based vaccines, lnt J Cancer. 2004 Nov 20; 112(4)669-77.

Zellweger T, Ninck C, Bloch M, Mirlacher M, Koivisto PA, Helin HJ, Mihatsch MJ, Gasser TC, Bubendorf L. Expression patterns of potential therapeutic targets in prostate cancer, lnt J Cancer. 2005 Feb 10; 113(4)619-28.

Nochi T, Yuki Y, Terahara K, Hino A, Kunisawa J, Kweon MN, Yamaguchi T, Kiyono H. Biological role of Ep-CAM in the physical interaction between epithelial cells and lymphocytes in intestinal epithelium. Clin Immunol. 2004 Dec; 113(3)326-39.

Tajima K, Demachi A₁ lto Y, Nishida K, Akatsuka Y, Tsujimura K, Kuwano H, Mitsudomi T, Takahashi T, Kuzushima K. Identification of an epitope from the epithelial cell adhesion molecule eliciting HLA-A*2402-restricted cytotoxic T-lymphocyte responses. Tissue Antigens. 2004 Dec;64(6)650-9.

Krishnakumar S, Mohan A, Mallikarjuna K, Venkatesan N, Biswas J, Shanmugam MP, Ren- Heidenreich L. EpCAM expression in retinoblastoma a novel molecular target for therapy. Invest Ophthalmol Vis ScL 2004 Dec;45(12)4247-50.

Haghighi KS, Woon WW, Akhter J, Marr PJ, Bolton E, Riordan S, Morris DL. A new source of hepatocytes for transplantation. Transplant Proc. 2004 Oct;36(8)2466-8.

Spizzo G, Went P, Dirnhofer S, Obrist P, Simon R, Spichtin H, Maurer R, Metzger U, von Castelberg B, Bart R, Stopatschinskaya S, Kochli OR, Haas P, Mross F, Zuber M, Dietrich H, Bischoff S, Mirlacher M, Sauter G, Gastl G. High Ep-CAM expression is associated with poor prognosis in node-positive breast cancer. Breast Cancer Res Treat. 2004 Aug;86(3)207-13.

Gutzmer R, Li W, Sutterwala S, Lemos MP, Elizalde Jl, Urtishak SL, Behrens EM, Rivers PM, Schlienger K, Laufer TM, Eck SL, Marks MS. A tumor-associated glycoprotein that blocks MHC class ll-dependent antigen presentation by dendritic cells. J Immunol. 2004 Ju1 15; 173(2): 1023-32. de Bono JS, Tolcher AW, Forero A, Vanhove GF, Takimoto C, Bauer RJ, Hammond LA, Patnaik A, White ML, Shen S, Khazaeli MB, Rowinsky EK, LoBuglio AF. ING-1 , a monoclonal antibody targeting Ep-CAM in patients with advanced adenocarcinomas. Clin Cancer Res. 2004 Nov 15;10(22)7555-65.

Mosolits S, Markovic K, Fagerberg J, Frodin JE, Rezvany MR, Kiaii S, Mellstedt H, Jeddi-Tehrani M. T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-id iotypic antibody. Cancer Immunol Immunother. 2005 Jun;54(6)557-70. Epub 2004 Nov 27.

Zbar AP. The immunology of colorectal cancer. Surg Oncol. 2004 Aug-Nov; 13(2-3)45-53. Review.

Xie X, Wang CY, Cao YX, Wang W, Zhuang R, Chen LH, Dang NN, Fang L, Jin BQ. Expression pattern of epithelial cell adhesion molecule on normal and malignant colon tissues. World J Gastroenterol. 2005 Jan 21 ;11(3)344-7.

Xie X, Wang CY, Cao YX, Wang W, Zhuang R, Chen LH, Dang NN, Fang L, Jin BQ. Expression pattern of epithelial cell adhesion molecule on normal and malignant colon tissues. World J Gastroenterol. 2005 Jan 21 ;11(3)344-7.

Prang N, Preithner S, Brischwein K, Goster P, Woppel A, Muller J, Steiger C, Peters M, Baeuerle PA, da Silva AJ. Cellular and complement-dependent cytotoxicity of Ep-CAM-specific monoclonal antibody MT201 against breast cancer cell lines. Br J Cancer. 2005 Jan 31 ;92(2)342-9.

Nagorsen D, Scheibenbogen C, Letsch A, Germer CT, Buhr HJ, Hegewisch-Becker S, Rivoltini L, Thiel E, Keilholz U. T cell responses against tumor associated antigens and prognosis in colorectal cancer patients. J Transl Med. 2005 Jan 19;3(1)3.

Crist KA, Zhang Z, You M, Gunning WT, Conran PB, Steele VE, Lubet RA. Characterization of rat ovarian adenocarcinomas developed in response to direct instillation of 7,12- dimethylbenz[a]anthracene (DMBA) coated suture. Carcinogenesis. 2005 May;26(5)951-7. Epub 2005 Feb 3.

Schlereth B, Fichtner I, Lorenczewski G, Kleindienst P, Brischwein K, da Silva A, Kufer P, Lutterbuese R, Junghahn I, Kasimir-Bauer S, Wimberger P, Kimmig R, Baeuerle PA. Eradication of tumors from a human colon cancer cell line and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific antibody construct. Cancer Res. 2005 Apr,1;65(7)2882-9.

Karanikiotis C, Skiadas I, Karina M, Georgakopoulou S, Georgakopoulos E, Fountzilas G. A novel chromatographic method for Ep-CAM mRNA detection in peripheral blood and bone marrow of patients with metastatic colorectal cancer. Anticancer Res. 2005 Jan-Feb;25(1A)319-23.

Songun I₁ Litvinov SV, van de Velde CJ, Pals ST, Hermans J, van Krieken JH. Loss of Ep-CAM (CO17-1A) expression predicts survival in patients with gastric cancer. Br J Cancer. 2005 May 9;92(9)1767-72.

Joo M, Kim H, Kim MK, Yu HJ, Kim JP. Expression of Ep-CAM in intestinal metaplasia, gastric epithelial dysplasia and gastric adenocarcinoma. J Gastroenterol Hepatol. 2005 Jul;20(7)1039-45.

Luborsky JL, Barua A, Shatavi SV, Kebede T, Abramowicz J, Rotmensch J.

Anti-tumor antibodies in ovarian cancer. Am J Reprod Immunol. 2005 Aug;54(2):55-62. Review.

Passebosc-Faure K, Li G, Lambert C, Cottier M, Gentil-Perret A, Fournel P, Perol M, Genin C. Evaluation of a panel of molecular markers for the diagnosis of Zalignant serous effusions. Clin Cancer Res. 2005 Oct 1 ;11 (19 Pt 1):6862-7.

Kuroki M, Yamada H, Shibaguchi H, Hachimine K, Hirose Y, Kinugasa T, lshida I₁ Kuroki M. Preparation of human IgG and IgM monoclonal antibodies for MK-1 /Ep-CAM by using human immunoglobulin gene-transferred mouse and gene cloning of their variable regions. Anticancer Res. 2005 Nov-Dec;25(6A):3733-9.

Breuhahn K, Baeuerle PA, Peters M, Prang N, Tox U, Kohne-Volland R, Dries V, Schirmacher P, Leo E.

Expression of epithelial cellular adhesion molecule (Ep-CAM) in chronic (necro-)inflammatory liver diseases and hepatocellular carcinoma. Hepatol Res. 2005 Dec 16; [Epub ahead of print]

Went P, Vasei M, Bubendorf L, Terracciano L, Tornillo L, Riede U, Kononen J, Simon R, Sauter G, Baeuerle PA.

Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers. Br J Cancer. 2006 Jan 16;94(1):128-35. Offner S, Hofmeister R, Romaniuk A, Kufer P, Baeuerle PA.

Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on

MHC class l-negative tumor cells.

MoI Immunol. 2006 Feb;43(6):763-71. Epub 2005 Apr 26.

Gommans WM, van Eert SJ, McLaughlin PM, Harmsen MC, Yamamoto M, Curiel DT, Haisma HJ,

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Schanzer JM, Baeuerle PA, Dreier T, Kufer P.

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As described in the publications above, Ep-CAM is a 4OkDa transmembrane glycoprotein broadly expressed on the basolateral surface of normal epithelia. Ep-CAM acts as an epithelial cell adhesion molecule and is involved in regulation of cadherin adhesions, and, possibly, cell proliferation and invasion. During carcinogenesis Ep-CAM has been shown to be strongly upregulated and increasing amounts of Ep-CAM also correlated with lower life expectancy of cancer patients. It has also been demonstrated that Ep-CAM positive colon cancer cells can be eradicated upon binding to toxin- conjugated or radiolabeled anti-Ep-CAM antibodies, presumably through the internalization signal located on the cytoplasmic tail of Ep-CAM. Consequently, active and passive immunotherapeutics targeting Ep-CAM are under clinical development and review of efficacy and safety in treating various carcinomas. While high affinity anti-Ep- CAM antibodies have demonstrated promoting ADCC and CDC in tumor cells, toxicity issues have been prevalent with these antagonists due to the broad expression of Ep-CAM in normal tissues. The Ovr232v3 variant of Ep-CAM is expressed as a 48 IcDa glycoprotein in transfected cells and has a unique 76aa insertion (Gly26-Leul01). The insertion is at the N-terminal end after the 23 amino acid secretory signal peptide in the extracellular region which is thought to be important for the cell-cell adhesion function of Ep-CAM. Ovr232v3 retains the two EGF-like domains, cystine-poor region, transmembrane domain and 26 amino acid cytoplasmic region characteristic of Ep-CAM. The 76 amino acid region unique to Ovr232v3 contains an RGD integrin binding domain (Arg68-Asp68) and a Pro-Arg rich domain implicated in protein-protein interaction (Pro79-Arg81). See Ruoslahti E, and Pierschbacher MD., Arg-Gly-Asp: a versatile cell recognition signal, Cell, (1986) 28, 517-8; and Bedford, et al, A novel pro-Arg motif recognized by WW domains, JBC _J (2000) 275, 10359-69; respectively. Additional protein features of Ovr232v3 such as post translational modifications (PTMs), motifs, structural and functional domains, and immunogenic properties are listed in the table below. The table lists the Type of feature; Type of PTM, Motif, Domain; Functional annotation for the Feature, Database or Prediction describing the feature; Database ID; the feature Start amino acid; and the feature stop amino acid.

While Ep-CAM is broadly expressed in normal tissues, using real-time quantitative RT-PCR we demonstrated Ovr232v3 expression is more restricted and more cancer- specific. For example, Ep-CAM exhibits significant expression in normal colon tissue, whereas Ovr232v3 is largely undetected in such samples. Irrespective of differing expression patterns, cell surface biotinylation and immunofluorescence experiments with live cells confirmed that the exogenously-expressed Ovr232v3 predominantly localizes to the plasma membrane.

When exposed to stressed conditions, stable cell lines expressing Ep-CAM or Ovr232v3 generally show growth advantages over negative control cells. These results are in agreement with literature reports that overexpression of Ep-CAM enhances cell proliferation and promotes colony formation in transfected cells. Therefore, like Ep- CAM, Ovr232v3 has utility as a therapeutic target. Additionally, like Ep-CAM, Ovr232v3 is contemplated to be useful as a cancer vaccine. Alternatively, Ovr232v3 is contemplated to have utility as a membrane bound or soluble antagonist of Ep-CAM function. Taken together, the high cancer specificity, cell surface localization and tumor growth promoting ability, make Ovr232v3 a promising target for immunotherapy of various tumor types. Specifically, Ovr232v3 is an attractive anti-tumor target due to the more restricted expression in cancer tissue compared to Ep-CAM which is broadly expressed in normal tissues. The higher cancer specific expression of Ovr232v3 limits the toxicity of anti-Ovr232v3 antibodies against normal tissues which is problematic in anti- Ep-CAM antibodies. Anti-Ovr232v3 antibodies are useful in therapeutic settings alone or in combination with anti-Ep-CAM antibodies where Ovr232v3 and/or Ep-CAM are overexpressed.

The term "antibody" (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Preferably, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for [L and F isotypes. Each 6 L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.

The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI).

Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Teff and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 1-10-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. around about residues 24-34 (LI), 5056 (L2) and 89-97 (L3) in the VL, and around about 1-35 (HI), 50-65 (H2) and 95-102 (113) in the VH; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (U) in the VL, and 26-32 (HI), 53-55 (1-12) and 96-101 (H3) in the VH; Chothia and Leak J. MoI. Biol. 196:901-917 (1987)).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al, Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. MoL Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen- binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.

An "intact" antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see US patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen- binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen- binding activity and is still capable of cross-linking antigen. Fab¹ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of 8 Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

"Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

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

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

The term "amino acid sequence variant" refers to a polypeptide that has amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants of Ovr232v3 will possess at least about 70% homology with the native sequence Ovr232v3, preferably, at least about 80%, more preferably at least about 85%, even more preferably at least about 90% homology, and most preferably at least 95%. The amino acid sequence variants can possess substitutions, deletions, insertions and/or alterations due to allelic variation or Single Nucleotide Polymorphisms (SNPs) within the native nucleic acid sequence encoding the amino acid sequence.

Several definitions of SNPs exist. See, e.g., Brooks, 235 Gene 177-86 (1999). As used herein, the term "single nucleotide polymorphism" or "SNP" includes all single base variants, thus including nucleotide insertions and deletions in addition to single nucleotide substitutions and any resulting amino acid variants due to codon alteration. There are two types of nucleotide substitutions. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine for a pyrimidine, or vice versa.

Numerous methods exist for detecting SNPs within a nucleotide sequence. A review of many of these methods can be found in Landegren et ah, 8 Genome Res. 769-76 (1998). For example, a SNP in a genomic sample can be detected by preparing a Reduced Complexity Genome (RCG) from the genomic sample, then analyzing the RCG for the presence or absence of a SNP. See, e.g., WO 00/18960. Multiple SNPs in a population of target polynucleotides in parallel can 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 by restriction 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 or destroys a restriction enzyme cleavage site. SNPs can also be detected by direct sequencing of the nucleotide sequence of interest. In addition, numerous assays based on hybridization have also been developed to detect SNPs and mismatch distinction by polymerases and ligases. Several web sites provide information about SNPs including Ensembl (ensembl with the extension .org of the world wide web), Sanger Institute (sanger with the extension .ac. uk/genetics/exon/ of the world wide web), National Center for Biotechnology Information (NCBI) (ncbi with the extension .nlm.nih.gov/SNP/ of the world wide web), The SNP Consortium Ltd. (snp with the extension .cshl.org of the world wide web). The chromosomal locations for the compositions disclosed herein are provided below. In addition, one of ordinary skill in the art could perform a search against the genome or any of the databases cited above using BLAST to find the chromosomal location or locations of SNPs. Another a preferred method to find the genomic coordinates and associated SNPs would be to use the BLAT tool (genome.ucsc.edu, Kent et al. 2001, The Human Genome Browser at UCSC, Genome Research 996-1006 or Kent 2002 BLAT, The BLAST -Like Alignment Tool Genome Research, 1-9). All web sites above were accessed December 3, 2003.

Preferred amino acid sequence variants of Ovr232v3 are described in the table below. The nucleic acid and amino acid sequences of Ovr232v3 are disclosed in WO 2004/053079 which is incorporated by reference in its entirety. The polynucleotides encoding the amino acids of the present invention were analyzed and single nucleotide polymorphism (SNP) attributes were identified. Specifically identified were SNPs occurring the coding region of the nucleotide, the Alleles of the SNP, the nucleotide ambiguity code for the SNP, the position in the codon of the SNP if within the Open Reading Frame (1, 2, 3 or UTR for untranslated regions), and the SNP type (synonymous or non-synonymous to the protein translation). In addition to the attributes above, the SNP rs# ID for the NCBI SNP database (dbSNP) which is accessible at ncbi with the extension .nlm.nih.gov/SNP/ of the world wide web is referenced for each SNP. Additional single nucleotide polymorphism (SNP) information can be accessed at the databases listed above.

The table below includes the polynucleotide target, dbSNP rs# ID, Nucleic acid residue affected by the SNP (Polynucleotide), SNP alleles, Nucleotide ambiguity code, Condon Position of the SNP if within the ORF (1, 2,3 or UTR if not within ORF), and the SNP type (synonymous "syn" or non-synonymous "non-syn"), Amino acid residue affected by the SNP (AA Residue), and the Alternate amino acid residue.

In summary, nucleic acid residues 1379, 1772, and 1778 of Ovr232v3 may be T or C, G or A, or T or C, respectively. Likewise, amino acid residues 191 and 324 may be M or T, or S or L, respectively due to SNP events.

Variants of Ovr232v3 as described above and antibodies which bind to these variants are part of the invention described herein. The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one which can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcεRI. "Homology" is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. Sequence similarity may be measured by any common sequence analysis algorithm, such as GAP or BESTFIT or other variation Smith- Waterman alignment. See, T. F. Smith and M. S. Waterman, J. MoI. Biol. 147:195-197 (1981) and W.R. Pearson, Genomics 11:635-650 (1991).

"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechniann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593- 596 (1992). Examples of humanized anti-Ep-CAM antibodies are described in US

2005/0009097 Al; Naundorf S et al., Int J Cancer. 100(l):101-10 (2002); Xiang W et al., J Cancer Res Clin Oncol. 129(6):341-8 (2003); and Prang N et al., Br J Cancer. 92(2):342- 9 (2005).

As used herein, an anti-Ovr232v3 antibody that "internalizes" is one that is taken up by (i.e., enters) the cell upon binding to Ovr232v3 on a mammalian cell (i.e. cell surface Ovr232v3). The internalizing antibody will of course include antibody fragments, human or humanized antibody and antibody conjugate. For therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill an Ovr232 v3 -expressing cell, especially an Ovr232v3 -expressing cancer cell. Depending on the potency of the antibody or antibody conjugate, in some instances, the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds. For example, certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill the tumor cell.

Whether an anti-Ovr232v3 antibody internalizes upon binding Ovr232v3 on a mammalian cell can be determined by various assays including those described in the experimental examples below. For example, to test internalization in vivo, the test antibody is labeled and introduced into an animal known to have Ovr232v3 expressed on the surface of certain cells. The antibody can be radiolabeled or labeled with fluorescent or gold particles, for instance. Animals suitable for this assay include a mammal such as a NCR nude mouse that contains a human Ovr232v3-expressing tumor transplant or xenograft, or a mouse into which cells transfected with human Ovr232v3 have been introduced, or a transgenic mouse expressing the human Ovr232v3 transgene. Appropriate controls include animals that did not receive the test antibody or that received an unrelated antibody, and animals that received an antibody to another antigen on the cells of interest, which antibody is known to be internalized upon binding to the antigen. The antibody can be administered to the animal, e.g., by intravenous injection. At suitable time intervals, tissue sections of the animal can be prepared using known methods or as described in the experimental examples below, and analyzed by light microscopy or electron microscopy, for internalization as well as the location of the internalized antibody in the cell. For internalization in vitro, the cells can be incubated in tissue culture dishes in the presence or absence of the relevant antibodies added to the culture media and processed for microscopic analysis at desired time points. The presence of an internalized, labeled antibody in the cells can be directly visualized by microscopy or by autoradiography if radiolabeled antibody is used. Alternatively, in a quantitative biochemical assay, a population of cells comprising Ovr232v3-expressing cells are contacted in vitro or in vivo with a radiolabeled test antibody and the cells (if contacted in vivo, cells are then isolated after a suitable amount of time) are treated with a protease or subjected to an acid wash to remove uninternalized antibody on the cell surface. The cells are ground up and the amount of protease resistant, radioactive counts per minute (cpm) associated with each batch of cells is measured by passing the homogenate through a scintillation counter. Based on the known specific activity of the radiolabeled antibody, the number of antibody molecules internalized per cell can be deduced from the scintillation counts of the ground- up cells. Cells are "contacted" with antibody in vitro preferably in solution form such as by adding the cells to the cell culture media in the culture dish or flask and mixing the antibody well with the media to ensure uniform exposure of the cells to the antibody. Instead of adding to the culture media, the cells can be contacted with the test antibody in an isotonic solution such as PBS in a test tube for the desired time period. In vivo, the cells are contacted with antibody by any suitable method of administering the test antibody such as the methods of administration described below when administered to a patient.

The faster the rate of internalization of the antibody upon binding to the Ovr232v3- expressing cell in vivo, the faster the desired killing or growth inhibitory effect on the target Ovr232v3-expressing cell can be achieved, e.g., by a cytotoxic immunoconjugate. Preferably, the kinetics of internalization of the anti-Ovr232v3 antibodies are such that they favor rapid killing of the Ovr232v3 -expressing target cell. Therefore, it is desirable that the anti-Ovr232v3 antibody exhibit a rapid rate of internalization preferably, within 24 hours from administration of the antibody in vivo, more preferably within about 12 hours, even more preferably within about 30 minutes to 1 hour, and most preferably, within about 30 minutes. The present invention provides antibodies that internalize as fast as about 15 minutes from the time of introducing the anti-Ovr232v3 antibody in vivo. The antibody will preferably be internalized into the cell within a few hours upon binding to Ovr232v3 on the cell surface, preferably within 1 hour, even more preferably within 15-30 minutes. To determine if a test antibody can compete for binding to the same epitope as the epitope bound by the anti-Ovr232v3 antibodies of the present invention including the antibodies produced by the hybridomas deposited with the ATCC, a cross-blocking assay e.g., a competitive ELISA assay can be performed. In an exemplary competitive ELISA assay, Ovr232v3 -coated wells of a microtiter plate, or Ovr232v3-coated sepharose beads, are pre-incubated with or without candidate competing antibody and then a biotin-labeled anti-Ovr232v3 antibody of the invention is added. The amount of labeled anti-Ovr232v3 antibody bound to the Ovr232v3 antigen in the wells or on the beads is measured using avidin-peroxidase conjugate and appropriate substrate.

Alternatively, the anti-Ovr232v3 antibody can be labeled, e.g., with a radioactive or fluorescent label or some other detectable and measurable label. The amount of labeled anti-Ovr232v3 antibody that binds to the antigen will have an inverse correlation to 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-Ovr-110 antibody will be bound to the antigen-coated wells. A candidate competing antibody is considered an antibody that binds substantially to the same epitope or that competes for binding to the same epitope as an anti-Ovr232v3 antibody of the invention if the candidate competing antibody can block binding of the anti-Ovr232v3 antibody by at least 20%, preferably by at least 20-50%, even more preferably, by at least 50% as compared to a control performed in parallel in the absence of the candidate competing antibody (but may be in the presence of a known noncompeting antibody). It will be understood that variations of this assay can be performed to arrive at the same quantitative value.

An antibody having a "biological characteristic" of a designated antibody, such as any of the monoclonal antibodies Ovr232v3.Cl, Ovr232v3.C2, Ovr232v3.C3, Ovr232v3.C4, Ovr232v3.C5, Ovr232v3.C6, Ovr232v3.C7, Ovr232v3.C8, Ovr232v3.C9, Ovr232v3.C10, Ovr232v3.Cl l, Ovr232v3.C12, Ovr232v3.C13, Ovr232v3.C14, Ovr232v3.C15, Ovr232v3.C16, Ovr232v3.C17, Ovr232v3.C18, Ovr232v3.C19, Ovr232v3.C20, Ovr232v3.C21, Ovr232v3.C22, Ovr232v3.C23, Ovr232v3.C24, Ovr232v3.C25, Ovr232v3.C26, Ovr232v3.C27, Ovr232v3.C28, Ovr232v3.C29, Ovr232v3.C30, Ovr232v3.C31, Ovr232v3.C32, Ovr232v3.C33, Ovr232v3.C34, Ovr232v3.C35, Ovr232v3.C36, Ovr232v3.C37, Ovr232v3.C38, Ovr232v3.C39, Ovr232v3.C40, Ovr232v3.C41, Ovr232v3.C42, Ovr232v3.C43, Ovr232v3.C44, Ovr232v3.C45, Ovr232v3.C46, Ovr232v3.C47, Ovr232v3.C48, Ovr232v3.C49, Ovr232v3.C50, Ovr232v3.C51, Ovr232v3.C52, Ovr232v3.C53, Ovr232v3.C54, Ovr232v3.C55, Ovr232v3.C56, Ovr232v3.C57, Ovr232v3.C58, Ovr232v3.C59, Ovr232v3.C60, Ovr232v3.C61, Ovr232v3.C62, Ovr232v3.C63, Ovr232v3.C64, Ovr232v3.C65, Ovr232v3.C66, Ovr232v3.Dl, Ovr232v3.D2, Ovr232v3.D3, Ovr232v3.D4, Ovr232v3.D5, Ovr232v3.D6, Ovr232v3.D7, Ovr232v3.D8, Ovr232v3.D9, Ovr232v3.D10, Ovr232v3.Dl l, Ovr232v3.D12, Ovr232v3.D13, Ovr232v3.D14, Ovr232v3.D15, Ovr232v3.D16, Ovr232v3.D17, Ovr232v3.D18, Ovr232v3.D19, Ovr232v3.D20, Ovr232v3.D21, Ovr232v3.D22, Ovr232v3.D23, Ovr232v3.D24, Ovr232v3.D25, Ovr232v3.D26, Ovr232v3.D27, Ovr232v3.D28, Ovr232v3.D29, Ovr232v3.D30, Ovr232v3.D31, Ovr232v3.D32, Ovr232v3.D33, Ovr232v3.D34, Ovr232v3.D35, Ovr232v3.D36, Ovr232v3.D37, Ovr232v3.D38, Ovr232v3.D39, Ovr232v3.D40, Ovr232v3.D41, Ovr232v3.D42, Ovr232v3.D43, and Ovr232v3.D44, is one which possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies that bind to the same antigen, Ovr232v3.Cl, Ovr232v3.C2, Ovr232v3.C3, Ovr232v3.C4, Ovr232v3.C5, Ovr232v3.C6, Ovr232v3.C7, Ovr232v3.C8, Ovr232v3.C9, Ovr232v3.C10, Ovr232v3.Cl 1, Ovr232v3.C12, Ovr232v3.C13, Ovr232v3.C14, Ovr232v3.C15, Ovr232v3.C16, Ovr232v3.C17, Ovr232v3.C18₅ Ovr232v3.C19, Ovr232v3.C20, Ovr232v3.C21, Ovr232v3.C22, Ovr232v3.C23, Ovr232v3.C24, Ovr232v3.C25, Ovr232v3.C26, Ovr232v3.C27, Ovr232v3.C28, Ovr232v3.C29, Ovr232v3.C30, Ovr232v3.C31, Ovr232v3.C32, Ovr232v3.C33, Ovr232v3.C34, Ovr232v3.C35, Ovr232v3.C36, Ovr232v3.C37, Ovr232v3.C38, Ovr232v3.C39, Ovr232v3.C40, Ovr232v3.C41, Ovr232v3.C42, Ovr232v3.C43, Ovr232v3.C44, Ovr232v3.C45, Ovr232v3.C46, Ovr232v3.C47, Ovr232v3.C48, Ovr232v3.C49, Ovr232v3.C50, Ovr232v3.C51, Ovr232v3.C52, Ovr232v3.C53, Ovr232v3.C54, Ovr232v3.C55, Ovr232v3.C56, Ovr232v3.C57, Ovr232v3.C58, Ovr232v3.C59, Ovr232v3.C60, Ovr232v3.C61, Ovr232v3.C62, Ovr232v3.C63, Ovr232v3.C64, Ovr232v3.C65, Ovr232v3.C66, Ovr232v3.Dl, Ovr232v3.D2, Ovr232v3.D3, Ovr232v3.D4, Ovr232v3.D5, Ovr232v3.D6, Ovr232v3.D7, Ovr232v3.D8, Ovr232v3.D9, Ovr232v3.D10, Ovr232v3.Dll, Ovr232v3.D12, Ovr232v3.D13, Ovr232v3.D14, Ovr232v3.D15, Ovr232v3.D16, Ovr232v3.D17, Ovr232v3.D18, Ovr232v3.D19, Ovr232v3.D20, Ovr232v3.D21, Ovr232v3.D22, Ovr232v3.D23, Ovr232v3.D24, Ovr232v3.D25, Ovr232v3.D26, Ovr232v3.D27, Ovr232v3.D28, Ovr232v3.D29, Ovr232v3.D30, Ovr232v3.D31, Ovr232v3.D32, Ovr232v3.D33, Ovr232v3.D34, Ovr232v3.D35, Ovr232v3.D36, Ovr232v3.D37, Ovr232v3.D38, Ovr232v3.D39, Ovr232v3.D40, Ovr232v3.D41, Ovr232v3.D42, Ovr232v3.D43, and Ovr232v3.D44 will bind the same epitope as that bound by Ovr232v3.C 1 , Ovr232v3.C2, Ovr232v3.C3, Ovr232v3.C4, Ovr232v3.C5, Ovr232v3.C6, Ovr232v3.C7, Ovr232v3.C8, Ovr232v3.C9, Ovr232v3.C10, Ovr232v3.Cl l, Ovr232v3.C12, Ovr232v3.C13, Ovr232v3.C14, Ovr232v3.C15, Ovr232v3.C16, Ovr232v3.C17, Ovr232v3.C18, Ovr232v3.C19, Ovr232v3.C20, Ovr232v3.C21, Ovr232v3.C22, Ovr232v3.C23, Ovr232v3.C24, Ovr232v3.C25, Ovr232v3.C26, Ovr232v3.C27, Ovr232v3.C28, Ovr232v3.C29, Ovr232v3.C30, Ovr232v3.C31, Ovr232v3.C32, Ovr232v3.C33, Ovr232v3.C34, Ovr232v3.C35, Ovr232v3.C36, Ovr232v3.C37, Ovr232v3.C38, Ovr232v3.C39, Ovr232v3.C40, Ovr232v3.C41, Ovr232v3.C42, Ovr232v3.C43, Ovr232v3.C44, Ovr232v3.C45, Ovr232v3.C46, Ovr232v3.C47, Ovr232v3.C48, Ovr232v3.C49, Ovr232v3.C50, Ovr232v3.C51, Ovr232v3.C52, Ovr232v3.C53, Ovr232v3.C54, Ovr232v3.C55, Ovr232v3.C56, Ovr232v3.C57, Ovr232v3.C58, Ovr232v3.C59, Ovr232v3.C60, Ovr232v3.C61, Ovr232v3.C62, Ovr232v3.C63, Ovr232v3.C64, Ovr232v3.C65, Ovr232v3.C66, Ovr232v3.Dl, Ovr232v3.D2₅ Ovr232v3.D3, Ovr232v3.D4, Ovr232v3.D5, Ovr232v3.D6, Ovr232v3.D7, Ovr232v3.D8, Ovr232v3.D9, Ovr232v3.D10, Ovr232v3.Dl l, Ovr232v3.D12, Ovr232v3.D13, Ovr232v3.D14, Ovr232v3.D15, Ovr232v3.D16, Ovr232v3.D17, Ovr232v3.D18, Ovr232v3.D19, Ovr232v3.D20, Ovr232v3.D21, Ovr232v3.D22, Ovr232v3.D23, Ovr232v3.D24, Ovr232v3.D25, Ovr232v3.D26, Ovr232v3.D27, Ovr232v3.D28, Ovr232v3.D29, Ovr232v3.D30, Ovr232v3.D31, Ovr232v3.D32, Ovr232v3.D33, Ovr232v3.D34, Ovr232v3.D35, Ovr232v3.D36, Ovr232v3.D37, Ovr232v3.D38, Ovr232v3.D39, Ovr232v3.D40₅ Ovr232v3.D41, Ovr232v3.D42, Ovr232v3.D43, and Ovr232v3.D44 (e.g. which competes for binding or blocks binding of monoclonal antibody Ovr232v3.Cl, Ovr232v3.C2, Ovr232v3.C3, Ovr232v3.C4, Ovr232v3.C5, Ovr232v3.C6, Ovr232v3.C7, Ovr232v3.C8, Ovr232v3.C9, Ovr232v3.C10, Ovr232v3.Cll, Ovr232v3.C12, Ovr232v3.C13, Ovr232v3.C14, Ovr232v3.C15, Ovr232v3.C16, Ovr232v3.C17, Ovr232v3.C18, Ovr232v3.C19, Ovr232v3.C20, Ovr232v3.C21, Ovr232v3.C22, Ovr232v3.C23, Ovr232v3.C24, Ovr232v3.C25, Ovr232v3.C26, Ovr232v3.C27, Ovr232v3.C28, Ovr232v3.C29, Ovr232v3.C30, Ovr232v3.C31, Ovr232v3.C32, Ovr232v3.C33, Ovr232v3.C34₅ Ovr232v3.C35, Ovr232v3.C36, Ovr232v3.C37, Ovr232v3.C38, Ovr232v3.C39, Ovr232v3.C40, Ovr232v3.C41, Ovr232v3.C42, Ovr232v3.C43, Ovr232v3.C44, Ovr232v3.C45, Ovr232v3.C46, Ovr232v3.C47, Ovr232v3.C48, Ovr232v3.C49, Ovr232v3.C50, Ovr232v3.C51, Ovr232v3.C52, Ovr232v3.C53, Ovr232v3.C54, Ovr232v3.C55, Ovr232v3.C56, Ovr232v3.C57, Ovr232v3.C58, Ovr232v3.C59, Ovr232v3.C60, Ovr232v3.C61, Ovr232v3.C62, Ovr232v3.C63, Ovr232v3.C64, Ovr232v3.C65, Ovr232v3.C66, Ovr232v3.Dl, Ovr232v3.D2, Ovr232v3.D3, Ovr232v3.D4, Ovr232v3.D5, Ovr232v3.D6, Ovr232v3.D7, Ovr232v3.D8, Ovr232v3.D9, Ovr232v3.D10,

Ovr232v3.Dl l, Ovr232v3.D12, Ovr232v3.D13, Ovr232v3.D14, Ovr232v3.D15, Ovr232v3.D16, Ovr232v3.D17, Ovr232v3.D18, Ovr232v3.D19, Ovr232v3.D20, Ovr232v3.D21, Ovr232v3.D22, Ovr232v3.D23, Ovr232v3.D24, Ovr232v3.D25, Ovr232v3.D26, Ovr232v3.D27, Ovr232v3.D28, Ovr232v3.D29, Ovr232v3.D30, Ovr232v3.D31, Ovr232v3.D32, Ovr232v3.D33, Ovr232v3.D34, Ovr232v3.D35, Ovr232v3.D36, Ovr232v3.D37, Ovr232v3.D38, Ovr232v3.D39, Ovr232v3.D40, Ovr232v3.D41, Ovr232v3.D42, Ovr232v3.D43, and Ovr232v3.D44), be able to target an Ovr232 v3 -expressing tumor cell in vivo and may internalize upon binding to Ovr232v3 on a mammalian cell in vivo. Likewise, an antibody with the biological characteristic of the Ovr232v3.Cl, Ovr232v3.C2, Ovr232v3.C3, Ovr232v3.C4, Ovr232v3.C5, Ovr232v3.C6, Ovr232v3.C7, Ovr232v3.C8, Ovr232v3.C9, Ovr232v3.C10, Ovr232v3.Cl l, Ovr232v3.C12, Ovr232v3.C13, Ovr232v3.C14, Ovr232v3.C15, Ovr232v3.C16, Ovr232v3.C17, Ovr232v3.C18, Ovr232v3.C19, Ovr232v3.C20, Ovr232v3.C21, Ovr232v3.C22, Ovr232v3.C23, Ovr232v3.C24, Ovr232v3.C25, Ovr232v3.C26, Ovr232v3.C27, Ovr232v3.C28, Ovr232v3.C29, Ovr232v3.C30, Ovr232v3.C31, Ovr232v3.C32, Ovr232v3.C33, Ovr232v3.C34, Ovr232v3.C35, Ovr232v3.C36, Ovr232v3.C37, Ovr232v3.C38, Ovr232v3.C39, Ovr232v3.C40, Ovr232v3.C41, Ovr232v3.C42, Ovr232v3.C43, Ovr232v3.C44, Ovr232v3.C45, Ovr232v3.C46, Ovr232v3.C47, Ovr232v3.C48, Ovr232v3.C49, Ovr232v3.C50, Ovr232v3.C51, Ovr232v3.C52, Ovr232v3.C53, Ovr232v3.C54, Ovr232v3.C55, Ovr232v3.C56, Ovr232v3.C57, Ovr232v3.C58, Ovr232v3.C59, Ovr232v3.C60, Ovr232v3.C61, Ovr232v3.C62, Ovr232v3.C63, Ovr232v3.C64, Ovr232v3.C65, Ovr232v3.C66,

Ovr232v3.Dl, Ovr232v3.D2, Ovr232v3.D3, Ovr232v3.D4, Ovr232v3.D5, Ovr232v3.D6, Ovr232v3.D7, Ovr232v3.D8, Ovr232v3.D9, Ovr232v3.D10, Ovr232v3.Dl l, Ovr232v3.D12, Ovr232v3.D13, Ovr232v3.D14, Ovr232v3.D15, Ovr232v3.D16, Ovr232v3.D17, Ovr232v3.D18, Ovr232v3.D19, Ovr232v3.D20, Ovr232v3.D21, Ovr232v3.D22, Ovr232v3.D23, Ovr232v3.D24, Ovr232v3.D25, Ovr232v3.D26, Ovr232v3.D27, Ovr232v3.D28, Ovr232v3.D29, Ovr232v3.D30, Ovr232v3.D31, Ovr232v3.D32, Ovr232v3.D33, Ovr232v3.D34, Ovr232v3.D35, Ovr232v3.D36, Ovr232v3.D37, Ovr232v3.D38, Ovr232v3.D39, Ovr232v3.D40, Ovr232v3.D41, Ovr232v3.D42, Ovr232v3.D43, and Ovr232v3.D44 antibody will have the same epitope binding, targeting, internalizing, tumor growth inhibitory and cytotoxic properties of the antibody. The term "antagonist" antibody is used in the broadest sense, and includes an antibody that partially or fully blocks, inhibits, or neutralizes a biological activity of a native Ovr232v3 protein disclosed herein. Methods for identifying antagonists of an Ovr232v3 polypeptide may comprise contacting an Ovr232v3 polypeptide or a cell expressing Ovr232v3 on the cell surface, with a candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the Ovr232v3 polypeptide.

The term 'agonistic" antibody is used in the broadest sese, and includes an antibody the partially or fully promotes, activates, or increases biological activity of Ovr232v3. Additionally, an agonistic antibody may mimic an Ovr232v3 binding partner (e.g. receptor or ligand) wherein binding of the Ovr232v3 antibody has substantially the same effect on biologic activity of Ovr232v3 as binding of the binding partner. Methods for identifying agonists of an Ovr232v3 polypeptide may comprise contacting an Ovr232v3 polypeptide or a cell expressing Ovr232v3 on the cell surface, with a candidate agonistic antibody and measuring a detectable change in one or more biological activities normally associated with the Ovr232v3 polypeptide.

An "antibody that inhibits the growth of tumor cells expressing Ovr232v3" or a "growth inhibitory" antibody is one which binds to and results in measurable growth inhibition of cancer cells expressing or overexpressing Ovr232v3. Preferred growth inhibitory anti-Ovr232v3 antibodies inhibit growth of Ovr232 v3 -expressing tumor cells (e.g., ovarian, colon, prostate or lung cancer cells) by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g. from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 pg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. The antibody is growth inhibitory in vivo if administration of the anti-Ovr232v3 antibody at about 1 pg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days. An antibody which "induces apoptosis" is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA₅ cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apop^'totic bodies). The cell is usually one which overexpresses Ovr232v3. Preferably the cell is a tumor cell, e.g. an ovarian, colon, prostate, or lung cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cells in an annexin binding assay. Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: CIq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); complement dependent cytotoxicity (CDC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.

Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457- 92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126.330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer, of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

"Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g. from blood. "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202 : 163 ( 1996) may be performed.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastema, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small- cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of 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, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain , as well as head and neck cancer, and associated metastases.

A "Ovr232v3 -expressing cell" is a cell which expresses endogenous or transfected Ovr232v3 on the cell surface. A "Ovr232v3-expressing cancer" is a cancer comprising cells that have Ovr232v3 protein present on the cell surface. A "Ovr232v3-expressing cancer" produces sufficient levels of Ovr232v3 on the surface of cells thereof, such that an anti-Ovr232γ 3 antibody can bind thereto and have a therapeutic effect with respect to the cancer. A cancer which "overexpresses" Ovr232v3 is one which has significantly higher levels of Ovr232v3 at the cell surface thereof, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. Ovr232v3 overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the Ovr232v3 protein present on the surface of a cell (e.g. via an immunohistochemistry assay; FACS analysis). Alternatively, or additionally, one may measure levels of Ovr232v3-encoding nucleic acid or mRNA in the cell, e.g. via fluorescent in situ hybridization; (FISH; see W098/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study Ovr232v3 overexpression by measuring shed antigen in a biological fluid such as serum, e.g., using antibody-based assays (see also, e.g., U.S. Patent No. 4,933,294 issued June 12, 1990; W091/05264 published April 18, 1991; U.S. Patent 5,401,638 issued

March 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 to an antibody which is optionally labeled with a detectable label, e.g. a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g. by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. An Ovr232v3- expressing cancer includes ovarian, colon, prostate, or lung cancer.

A "mammal" for purposes of treating a cancer or alleviating the symptoms of cancer, refers to any mammal, including-humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human. "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for an Ovr232 v3 -expressing cancer if, after receiving a therapeutic amount of an anti-

Ovr232v3 antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the anti-Ovr232v3 antibody may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

The term "therapeutically effective amount" refers to an amount of an antibody or a drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See preceding definition of "treating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.

"Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.

Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such 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 as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², 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, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, e.g., gelonin, ricin, saporin, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially an Ovr232v3-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of Ovr232v3 -expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce GI arrest and M-phase arrest. Classical M- phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest GI also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, 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 from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

"Label" as used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

The term "epitope tagged" used herein refers to a chimeric polypeptide comprising an anti-Ovr232v3 antibody polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the Ig polypeptide to which it is fused. The tag polypeptide is also preferably fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

A "small molecule" is defined herein to have a molecular weight below about 500 Daltons.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

An "isolated nucleic acid molecule" is a nucleic acid molecule, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid molecule which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid molecule includes isolated forms of the nucleic acid molecule.

"Vector" includes shuttle and expression vectors and includes, e.g., a plasmid, cosmid, or phagemid. Typically, a plasmid construct will also include an origin of replication (e.g., the CoIEl origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively, of the plasmids in bacteria. An "expression vector" refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment of the invention, in prokaryotic, e.g., bacterial, or eukaryotic cells. Suitable vectors are disclosed below.

The cell that produces an anti-Ovr232v3 antibody of the invention will include the parent hybridoma cell e.g., the hybridomas that are deposited with the ATCC, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibodies have been introduced. Suitable host cells are disclosed below. RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2',5'- oligoadenylate synthetase resulting in non-specific cleavage of niRNA by ribonuclease L. The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. Elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21 -nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila 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 to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3 '-overhangs. Furthermore, complete substitution of one or both siRNA strands with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3 '-terminal siRNA overhang nucleotides with deoxy nucleotides (2'-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5'-end of the siRNA guide sequence rather than the 3'-end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated that a 5'-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5'-phosphate moiety on the siRNA (Nykanen et al., 2001,

Cell, 107, 309). -

Studies have shown that replacing the 3 '-overhanging segments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2'-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication No. WO 01/68836 both suggest that siRNA "may include modifications to either the phosphate-sugar back bone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom", however neither application teaches to what extent these modifications are tolerated in siRNA molecules nor provide any examples of such modified siRNA. Kreutzer and Limmer, Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double stranded-RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-O-methyl nucleotides, and nucleotides containing a 2'-0 or 4'-C methylene bridge. However, Kreutzer and Limmer similarly fail to show to what extent these modifications are tolerated in siRNA molecules nor do they provide any examples of such modified siRNA. Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that "RNAs with two (phosphorothioate) modified bases also had substantial decreases in effectiveness as RNAi triggers (data not shown);

(phosphorothioate) modification of more than two residues greatly destabilized the RNAs in vitro and we were not able to assay interference activities." Id. at 1081. The authors also tested certain modifications at the 2'-position of the nucleotide sugar in the long siRNA transcripts and observed that substituting deoxynucleotides for ribonucleotides "produced a substantial decrease in interference activity", especially in the case of Uridine to

Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting 4-thiouracil, 5-bromouracil, 5- iodouracil, 3-(aminoallyl)uracil for uracil, and inosine for guanosine in sense and antisense strands of the siRNA, and found that whereas 4-thiouracil and 5-bromouracil were all well tolerated, inosine "produced a substantial decrease in interference activity" when incorporated in either strand. Incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in substantial decrease in RNAi activity as well.

Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describes a Drosophila in vitro

RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due "to the danger of activating interferon response". Li et al., International PCT Publication No. WO 00/44914, describes the use of specific dsRNAs for use in attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describes certain methods for inhibiting the expression of particular genes in mammalian cells using certain dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describes particular methods for introducing certain dsRNA 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 genes responsible for conferring a particular phenotype in a cell using specific dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describes the identification of specific genes involved in dsRNA mediated RNAi. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describes specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Driscoll et al., International PCT Publication No. WO 01/49844, describes specific DNA constructs for use in facilitating gene silencing in targeted organisms. Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describes specific chemically modified siRNA constructs targeting the unc-22 gene of C. elegans. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs.

Compositions and Methods of the Invention The_.invention/provides anti-Ovr232v3 antibodies. -Preferably, the anti-Ovr232v3 antibodies internalize upon binding to cell surface Ovr232v3 on a mammalian cell. The anti-Ovr232v3 antibodies may also destroy or lead to the destruction of tumor cells bearing Ovr232v3.

It was not apparent that Ovr232v3 was internalization-competent. In addition the ability of an antibody to internalize depends on several factors including the affinity, avidity, and isotype of the antibody, and the epitope that it binds. We have demonstrated herein that the cell surface Ovr232v3 is internalization competent upon binding by the anti-Ovr232v3 antibodies of the invention. Additionally, it was demonstrated that the anti- Ovr232v3 antibodies of the present invention can specifically target Ovr232v3-expressing tumor cells. These tumor targeting, internalization and growth inhibitory properties of the anti-Ovr232v3 antibodies make these antibodies very suitable for therapeutic uses, e.g., in the treatment of various cancers including ovarian, colon, prostate, or lung cancer. Internalization of the anti-Ovr232v3 antibody is preferred, e.g., if the antibody or antibody conjugate has an intracellular site of action and if the cytotoxic agent conjugated to the antibody does not readily cross the plasma membrane (e.g., the toxin calicheamicin). Internalization is not necessary if the antibodies or the agent conjugated to the antibodies do not have intracellular sites of action, e.g., if the antibody can kill the tumor cell by ADCC or some other mechanism.

The anti-Ovr232v3 antibodies of the invention also have various non-therapeutic applications. The anti-Ovr232v3 antibodies of the present invention can be useful for diagnosis and staging of Ovr232v3-expressing cancers (e.g., in radioimaging). They may be used alone or in combination with other ovarian cancer markers, including, but not limited to, CA125, HE4 and mesothelin. The antibodies are also useful for purification or immunoprecipitation of Ovr232v3 from cells, for detection and quantitation of Ovr232v3 in vitro, e.g. in an ELISA or a Western blot, to kill and eliminate Ovr232 v3 -expressing cells from a population of mixed cells as a step in the purification of other cells. The internalizing anti-Ovr232v3 antibodies of the invention can be in the different forms encompassed by the definition of "antibody" herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In fusion antibodies, an antibody sequence is fused to a heterologous polypeptide sequence. The antibodies can be modified in the Fc region to provide desired . . effector functions. As discussed in more detail in the sections below, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used.

The antibody may compete for binding, or binds substantially to, the same epitope bound by the antibodies of the invention. Antibodies having the biological characteristics of the present anti-Ovr232v3 antibodies of the invention are also contemplated, e.g., an anti-Ovr232v3 antibody which has the biological characteristics of a monoclonal antibody produced by the hybridomas deposited with the ATCC on 29 July 2005 comprising Ovr232v3.C31.1 and Ovr232v3.C32.3, specifically including the in vivo tumor targeting, internalization and any cell proliferation inhibition or cytotoxic characteristics.

Specifically provided are anti-Ovr232v3 antibodies that 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 or 1-15, 10-25, 15-25, 21-35, 31-45, 41-55, 51-65, 61-75, 71-85, 81-95, 91-105, 101-115, 111-125, 121-135, 131-145, 141-155, 151-165, 161-175, 171-185, 181-195, 191-205, 201- 215, 211-225, 221-235, 231-245, 241-255, 251-265, 261-275, 271-285, 281-295, 291-305, 301-315, 311-325, 321-335, 331-345, 341-355, 351-365, 361-375, 371-385, 381-390 or 25-35, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, 70-80, 75-85, 80-90, 85- 95, 90-100, 95-101 of human Ovr232v3. Methods of producing the above antibodies are described in detail below.

The present anti-Ovr232v3 antibodies are useful for treating an Ovr232v3- expressing cancer or alleviating one or more symptoms of the cancer in a mammal. Such a cancer includes ovarian, colon, prostate, or lung cancer, cancer of the urinary tract, lung cancer, breast cancer, colon cancer, pancreatic cancer, and ovarian cancer, more specifically, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma. The cancers encompass metastatic cancers of any of the preceding, e.g., ovarian, colon, prostate, or lung cancer metastases. The antibody is able to bind to at least a portion of the cancer cells that express Ovr232v3 in the mammal and preferably is one that does not induce or that minimizes HAMA response. Preferably, the antibody is effective to destroy or kill Ovr232v3-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to Ovr232v3 on the cell. Such an antibody includes a naked anti- Ovr232v3 antibody (not conjugated to any agent). Naked anti-Ovr232v3 antibodies having tumor growth inhibition properties in vivo include the antibodies described in the Experimental Examples below. Naked antibodies that have cytotoxic or cell growth inhibition properties can be further conjugated with a cytotoxic agent to render them even more potent in tumor cell destruction. Cytotoxic properties can be conferred to an anti- Ovr232v3 antibody by, e.g., conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described below. The cytotoxic agent or a growth inhibitory agent is preferably a small molecule. Toxins such as maytansin, maytansinoids, saporin, gelonin, ricin or calicheamicin and analogs or derivatives thereof, are preferable. The invention provides a composition comprising an anti-Ovr232v3 antibody of the invention, and a carrier. For the purposes of treating cancer, compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-Ovr232v3 antibodies present as an immunoconjugate or as the naked antibody. Further, the compositions can comprise these antibodies in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides formulations comprising an anti- Ovr232v3 antibody of the invention, and a carrier. The formulation may be a therapeutic formulation comprising a pharmaceutically acceptable carrier. Another aspect of the invention is isolated nucleic acids encoding the internalizing anti-Ovr232v3 antibodies. Nucleic acids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are encompassed. The invention also provides methods useful for treating an Ovr232v3 -expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an internalizing anti-Ovr232v3 antibody to the mammal. The antibody therapeutic compositions can be administered short term (acute) or chronic, or intermittent as directed by physician. Also provided are methods of inhibiting the growth of, and killing an Ovr232v3 expressing cell. Finally, the invention also provides kits and articles of manufacture comprising at least one antibody of this invention, preferably at least one internalizing anti-Ovr232v3 antibody of this invention. Kits containing anti-Ovr232v3 antibodies find use in detecting Ovr-110 expression, or in therapeutic or diagnostic assays, e.g., for Ovr232v3 cell killing assays or for purification and/or immunoprecipitation of Ovr232v3 from cells. For example, for isolation and purification of Ovr232v3, the kit can contain an anti-Ovr232v3 antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain antibodies for detection and quantitation of Ovr232v3 in vitro, e.g. in an ELISA or a Western blot. Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel. Production of anti-Oyr232v3 antibodies

The following describes exemplary techniques for the production of the antibodies useful in the present invention. Some of these techniques are described further in Example 1. The Ovr232v3 antigen to be used for production of antibodies may be, e.g., the full length polypeptide or a portion thereof, including a soluble form of Ovr232v3 lacking the membrane spanning sequence, or synthetic peptides to selected portions of the protein.

Alternatively, cells expressing Ovr232v3 at their cell surface (e.g. CHO or NIH- 3T3 cells transformed to overexpress Ovr232v3; ovarian, pancreatic, lung, breast or other Ovr232v3-expressing tumor cell line), or membranes prepared from such cells can be used to generate antibodies. The nucleotide and amino acid sequences of human and murine Ovr232v3 are available as provided above. Ovr232v3 can be produced recombinantly in and isolated from, prokaryotic cells, e.g., bacterial cells, or eukaryotic cells using standard recombinant DNA methodology. Ovr232v3 can be expressed as a tagged (e.g., epitope tag) or other fusion protein to facilitate its isolation as well as its identification in various assays.

Antibodies or binding proteins that bind to various tags and fusion sequences are available as elaborated below. Other forms of Ovr232v3 useful for generating antibodies will be apparent to those skilled in the art.

Tags

Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 (Field et al., MoI. Cell. Biol, 8:2159- 2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990)). The FLAG-peptide (Hopp et al., BioTechnology, 6:1204-1210 (1988)) is recognized by an anti-FLAG M2 monoclonal antibody (Eastman Kodak Co., New Haven, CT). Purification of a protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently attached to agarose (Eastman Kodak Co., New Haven, CT). Other tag polypeptides include the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide (Skinner et al., J. Biol. Chenz., 266:15163-15166 (1991)); and the T7 gene protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)). Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals, preferably non-human animals, by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N=C=NR, where R and R¹ are different alkyl groups. Conjugates also can be made in recombinant cell culture as protein fusions.

Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 5-100 pg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Also,, aggregating agents such as alum are suitably used to enhance the immune response. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a "fusion partner", e.g., a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies. Principles and Practice, pp 103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, fusion partner, e.g., the parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-II mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, California US A, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications _vpp. 51_^63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980). Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed or transfected into prokaryotic or eukaryotic host cells such as, e.g., E coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Phickthun, Immunol. Revs., 130:151-188 (1992).

Further, the monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol., 222:581-597 (1991) describe the isolation of murine 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 combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding W

69

sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The nonimmunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

Humanized Antibodies

Methods for humanizing non-human antibodies have been described in the ait. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following 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)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. MoI. Biol, 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol, 151:2623 (1993)). It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Various forms of a humanized anti-Ovr232v3 antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgGl antibody.

Human Antibodies As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J_H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies 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. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and, alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimniunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al.,

Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the. spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. MoI. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Patent Nos. 5,565,332 and 5,573,905. As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275).

Antibody Fragments In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab)2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. The antibody of choice may also be a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific. . .. ._ - - ^■- -

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Ovr232v3 protein. Other such antibodies may combine an Ovr232v3 binding site with a binding site for another protein. Alternatively, an anti-Ovr232v3.Arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a Tcell receptor molecule (e.g. C 133), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRlI (CD32) and FcγRIII (CD 16), so as to focus and localize cellular defense mechanisms to the Ovr232v3-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Ovr232v3. These antibodies possess an Ovr232v3- binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab)2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein etal, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, EMBO J., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) 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_H2, and C_H3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the. site necessary for light-chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.

Preferably, the bispecific antibodies in this approach are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecifϊc molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121:210 (1986). According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175 : 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

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

The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making 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).

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

Multivalent Antibodies A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VDl(X In- VD2-(X2)n-Fc, wherein VDI is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CHI-flexible linker- VH-CHI-Fc region chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the anti-Ovr232v3 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the anti-Ovr232v3 antibody are prepared by introducing appropriate nucleotide changes into the anti-Ovr232v3 antibody nucleic acid, or by peptide synthesis.

Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the anti-Ovr232v3 antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the anti-Ovr232v3 antibody, such as changing the number or position of glycosylation sites. A useful method for identification of certain residues or regions of the anti- Ovr232v3 antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells in Science, 244:1081-1085 (1989). Here, a residue or group of target residues within the anti-Ovr232v3 antibody are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with Ovr232v3 antigen.

Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at a target codon or region and the expressed anti-Ovr232v3 antibody variants are screened for the desired activity. Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acidresidues. Examples of terminal insertions include an anti-Ovr232v3 antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the anti-Ovr232v3 antibody molecule include the fusion to the N- or C-terminus of the anti- Ovr232v3 antibody to an enzyme (e.g. for ADEPT) or a fusion to a polypeptide which increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-Ovr232v3 antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table I under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened for a desired characteristic.

TABLE I Amino Acid Substitutions

Substantial modifications in the biological properties of the antibody are accomplished ^"by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, 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 are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: 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.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the anti-Ovr232v3 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human Ovr232v3. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked or O-linked. N- linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of the anti-

Ovr232v3 antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid molecule encoding a variant or a non- variant version of the anti-

Ovr232v3 antibody. It may be desirable to modify the antibody of the invention with respect to effector function, e.g. so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.

Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell 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 with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer

Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities.

See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of the antibody.

Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One may further select antibodies with certain biological characteristics, as desired.

The growth inhibitory effects of an anti-Ovr232v3 antibody of the invention may be assessed by methods known in the art, e.g., using cells which express Ovr232v3 either endogenously or following transfectioii with the Ovr232v3 gene. For example, the tumor cell lines and Ovr232v3-transfected cells provided in Example 1 below may be treated with an anti-Ovr232v3 monoclonal antibody of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing H-thymidine uptake by the cells treated in the presence or absence an anti- Ovr232v3 antibody of the invention. After antibody treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriated positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. Preferably, the tumor cell is one that over- expresses Ovr232v3. Preferably, the anti-Ovr232v3 antibody will inhibit cell proliferation of an Ovr232v3-expressing tumor cell in vitro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, at an antibody concentration of about 0.5 to 30 μg/ml. Growth inhibition can be measured at an antibody concentration ofabout 0.5. to 30 μg/ml - or about 0.5 nM to 20OnM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of the anti-Ovr232v3 antibody at about lμg/kg to about

100mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

To select for antibodies which induce cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to a control. A PI uptake assay can be performed in the absence of complement and immune effector cells. Ovr232 v3 -expressing tumor cells are incubated with medium alone or medium containing of the appropriate monoclonal antibody at e.g., about lOμg/ml. The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 mm strainer-capped 12 x 75 tubes (ImI per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (lOμg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Those antibodies which induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death- inducing antibodies.

To screen for antibodies which bind to an epitope on Ovr232v3 bound by an antibody of interest, e.g., the Ovr232v3 antibodies of this invention, a routine cross- blocking assay such as that describe in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody binds the same site or epitope as an anti-Ovr232v3 antibody of the invention. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initially tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of Ovr232v3 can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

For example, a method to screen for antibodies that bind to an epitope which is bound by an antibody this invention may comprise combining an Ovr23.2 v3 -containing sample with a test antibody and an antibody of this invention to form a mixture , the level of Ovr232v3 antibody bound to Ovr232v3 in the mixture is then determined and compared to the level of Ovr232v3 antibody bound in the mixture to a control mixture, wherein the level of Ovr232v3 antibody binding to Ovr232v3 in the mixture as compared to the control is indicative of the test antibody's binding to an epitope that is bound by the anti- Ovr232v3 antibody of this invention. The level of Ovr232v3 antibody bound to Ovr232v3 is determined by ELISA. The control may be a positive or negative control or both. For example, the control may be a mixture of Ovr232v3, Ovr232v3 antibody of this invention and an antibody known to bind the epitope bound by the Ovr232v3 antibody of this invention. The anti-Ovr232v3 antibody labeled with a label such as those disclosed herein. The Ovr232v3 may be bound to a solid support, e.g., a tissue culture plate or to beads, e.g., sepharose beads. Immunoconjugates

The invention also pertains to therapy with immunoconjugates comprising an antibody conjugated to an anti-cancer agent such as a cytotoxic agent or a growth inhibitory agent. Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CCl 065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.

Maytansine and maytansinoids Preferably, an anti-Ovr232v3 antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization.

Maytansine was first isolated from the cast African shrub Maytenus serrata (U.S. Patent

No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No.

4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent 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, the disclosures of which are hereby expressly incorporated by reference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DMI linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al. Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA.l-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 x 10 5 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansonid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7- maytansinoid conjugate showed low systemic cytotoxicity in mice.

Anti-Ovr232v3 antϊbody-Maytansinoid Conjugates (Immunoconjugates) Anti-Ovr232v3 antibody-maytansinoid conjugates are prepared by chemically linking an anti-Ovr232v3 antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.

There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or EP Patent 0425 235 B 1, and Chari et al. Cancer Research 52: 127-131 (1992). The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred. Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl (2-pyridyidithio) propionate (SPDP), succinimidyl- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as his (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6diisocyanate), and bis-active fluorine compounds (such as l,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.

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

Calicheamicin Another immunoconjugate of interest comprises an anti-Ovr232v3 antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. patents 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, γ/, Ot₂ ¹, Ct₃ ¹, N-acetyl- γi¹, PSAG and O₁ ¹, (Hinman et al. Cancer Research 53: 3336 (1993), Lode et al. Cancer Research 5 8: 2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-Ovr232v3 antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. patents 5,053,394, 5,770,710, as well as esperamicins (U.S. patent 5,877,296). Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, 1 5 nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantliin proteins, Pliytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993. The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase). For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-Ovr232v3 antibodies. Examples include At²¹¹, 1¹³¹, 1¹²⁵, In¹¹ ^Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example Tc^99M or I¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123,jodine-131,indium-l 11, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen. Labels such as Tc^99M, I¹²³, In¹¹¹, Re¹⁸⁶, Re¹⁸⁸, can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon labeled 1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfϊde-containing linker (Chari et al. Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-Ovr232v3 antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

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

Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see W081/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Patent No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form. Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic fluorocytosine into the anticancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as O-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; P-lactamase useful for converting drugs derivatized with P- lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens 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 the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population. The enzymes of this invention can be covalently bound to the anti-Ovr232v3 antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well, known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984).

Other Antibody Modifications Other modifications of the antibody are contemplated herein. For example, the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The anti-Ovr232v3 antibodies disclosed herein may also be formulated as irnmunoliposom.es. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods 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 and 4,544,545; and W097/38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst.81(19)1484 (1989). Vectors, Host Cells, and Recombinant Methods

The invention also provides isolated nucleic acid molecule encoding the humanized anti-Ovr232v3 antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody. For recombinant production of the antibody, the nucleic acid molecule encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or inserted into a vector in operable linkage with a promoter for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to nucleic acid molecules encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

Signal Sequence Component

The anti-Ovr232v3 antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N- terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native anti- Ovr232v3 antibody signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, oc factor leader (including Saccharomyces and Kluyveromyces cc-factor leaders), or acid phosphatase leader, the C albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such precursor region is ligated in reading frame to DNA encoding the anti-Ovr232v3 antibody.

Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA; and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the S V40 origin may typically be used only because it contains the early promoter).

Selection Gene Component Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-Ovr232v3 antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -11, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL- 9096).

Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding anti-Ovr232v3 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature, 282:39 (1979)). The trpl gene provides a selection marker for a mutant 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 tipl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-defϊcient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDI can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large- scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991). Promoter Component

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-Ovr232v3 antibody nucleic acid. Promoters suitable for use with prokaryotic hosts include the phoA promoter , P-lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine- Dalgarno (S. D.) sequence operably linked to the DNA encoding the anti-Ovr232v3 . antibody. Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors. Examples of suitable promoter sequences for Use with yeast hosts include the promoters for 3 -phosphogly cerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.

Anti-Ovr232v3 antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma vims as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human P-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Enhancer Element Component

Transcription of a DNA encoding the anti-Ovr232v3 antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, arid insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the anti- Ovr232v3 antibody-encoding sequence, but is preferably located at a site 5' from the promoter. TranscriptionTermination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-Ovr232v3 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vector disclosed therein.

Selection and Transformation of Host Cells Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli Xl 776 (ATCC 31,537), and E. coli W31 10 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. 5,648,237 (Carter et. al), U.S. 5,789,199 (JoIy et al), and U.S. 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 soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g,, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-Ovr232v3 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts 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 (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-Ovr232v3 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-I variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

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

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)) ; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cellsADHFR (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 CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA₅ ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 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).

Host cells are transformed with the above-described expression or cloning vectors for anti-Ovr232v3 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Culturing Host Cells

The host cells used to produce the anti-Ovr232v3 antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's FIO (Sigma), Minimal Essential Medium (MEM)(Sigma), RPMI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM)(Sigma) are suitable for culturing the host cells. In addition, 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. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented 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, and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Purification ofanti-Ovr232v3 antibody

When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which 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 is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γl, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SIDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 - 4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt). Pharmaceutical Formulations

Pharmaceutical formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and mcresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyllolidone; 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; tόnicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml.

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to the anti- Ovr232v3 antibody which internalizes, it may be desirable to include in the one formulation, an additional antibody, e.g. a second anti-Ovr232v3 antibody which binds a different epitope on Ovr232v3, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or 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. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-) hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Methods and Treatment Using Anti-Oyr232v3 Antibodies According to the present invention, the anti-Ovr232v3 antibody that internalizes upon binding Ovr232v3 on a cell surface is used to treat a subject in need thereof having a cancer characterized by Ovr232v3 -expressing cancer cells, in particular, ovarian, colon, prostate, or lung cancer, and associated metastases.

The cancer will generally comprise Ovr232v3-expressing cells, such that the anti- Ovr232v3 antibody is able to bind thereto. While the cancer may be characterized by overexpression of the Ovr232v3 molecule, the present application further provides a method for treating cancer which is not considered to be an Ovr232v3-overexpressing cancer.

This invention also relates to methods for detecting cells or tissues which overexpress Ovr232v3 and to diagnostic kits useful in detecting cells or tissues expressing Ovr232v3 or in detecting Ovr232v3 in bodily fluids from a patient. Bodily fluids include blood, serum, plasma, urine, ascites, peritoneal wash, saliva, sputum, seminal fluids, mucous membrane secretions, and other bodily excretions such as stool. The methods may comprise combining a cell-containing test sample with an antibody of this invention, assaying the test sample for antibody binding to cells in the test sample and comparing the level of antibody binding in the test sample to the level of antibody binding in a control sample of cells. A suitable control is, e.g., a sample of normal cells of the same type as the test sample or a cell sample known to be free of Ovr232v3 overexpressing cells. A level of Ovr232v3 binding higher than that of such a control sample would be indicative of the test sample containing cells that overexpress Ovr232v3. Alternatively the control may be a sample of cells known to contain cells that overexpress Ovr232v3. hi such a case, a level of Ovr232v3 antibody binding in the test sample that is similar to, or in excess of, that of the control sample would be indicative of the test sample containing cells that overexpress Ovr232v3.

Ovr232v3 overexpression may be detected with a various diagnostic assays. For example, over expression of Ovr232v3 may be assayed by immunohistochemistry (IHC). Paraffin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded an Ovr232v3 protein staining intensity criteria as follows.

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

Score 1+ a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane.

Score 2+ a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.

Score 3+ a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells. Those tumors with 0 or 1+ scores for Ovr232v3 expression may be characterized as not overexpressing Ovr232v3, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing Ovr232v3.

Alternatively, or additionally, FISH assays such as the INFORM™ (sold by Ventana, Arizona) or PATHVISION™ (VySiS, Illinois) may be carried out on formalin- fixed, paraffin-embedded tumor tissue to determine the extent (if any) of Ovr232v3 overexpression in the tumor. Ovr232v3 overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody of this invention) which binds Ovr232v3 and which is labeled with a detectable label (e.g. a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.

A sample suspected of containing cells expressing or overexpressing Ovr232v3 is combined with the antibodies of this invention under conditions suitable for the specific binding of the antibodies to Ovr232v3. Binding and/or internalizing the Ovr232v3 antibodies of this invention is indicative of the cells expressing Ovr232v3. The level of binding may be determined and compared to a suitable control, wherein an elevated level of bound Ovr232v3 as compared to the control is indicative of Ovr232v3 overexpression. The sample suspected of containing cells overexpressing Ovr232v3 may be a cancer cell sample, particularly a sample of ovarian, colon, prostate or lung cancer. A serum sample from a subject may also be assayed for levels of Ovr232v3 by combining a serum sample from a subject with an Ovr232v3 antibody of this invention, determining the level of Ovr232v3 bound to the antibody and comparing the level to a control, wherein an elevated level of Ovr232v3 in the serum of the patient as compared to a control is indicative of overexpression of Ovr232v3 by cells in the patient. The subject may have a cancer such as ovarian, colon, prostate or lung cancer.

Currently, depending on the stage of the cancer, ovarian, colon, prostate, or lung cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, androgen deprivation (e.g., hormonal therapy), and chemotherapy. Anti-Ovr232v3 antibody therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well, in metastatic disease where radiation therapy has limited usefulness, and for the management of prostatic carcinoma that is resistant to androgen deprivation treatment. The tumor targeting and internalizing anti-Ovr232v3 antibodies of the invention are useful to alleviate Ovr232 v3 -expressing cancers, e.g., ovarian, colon, prostate, or lung cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, the anti-Ovr232v3 antibody can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabeled compounds, or with surgery, cryotherapy, and/or radiotherapy, notably for ovarian, colon, prostate, or lung cancers, also particularly where shed cells cannot be reached. Anti-Ovr232v3 antibody treatment can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy, Chemotherapeutic drugs such as Taxotere® (docetaxel), Taxol® (paclitaxel), estramustine and mitoxantrone are used in treating metastatic and hormone refractory ovarian, colon, prostate, or lung cancer, in particular, in good risk patients. In the present method of the invention for treating or alleviating cancer, in particular, androgen independent and/or metastatic ovarian, colon, prostate, or lung cancer, the cancer patient can be administered anti-Ovr232v3 antibody in conjunction with treatment with the one or more of the preceding chemotherapeutic agents. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EP0600517) is contemplated. The anti-Ovr232v3 antibody will be administered with a therapeutically effective dose of the chemotherapeutic agent. The anti-Ovr232v3 antibody may also be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages of these agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.

Particularly, an immunoconjugate comprising the anti-Ovr232v3 antibody conjugated with a cytotoxic agent may be administered to the patient. Preferably, the immunoconjugate bound to the Ovr232v3 protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. Preferably, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents are described above and include maytansin, maytansinoids, saporin, gelonin, ricin, calicheamicin, ribonucleases and DNA endonucleases.

The anti-Ovi'232v3 antibodies or immunoconjugates are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The antibodies or immunoconjugates may be injected directly into the tumor mass. Intravenous or subcutaneous administration of the antibody is preferred. Other therapeutic regimens may be combined with the administration of the anti- Ovr232v3 antibody.

The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.

It may also be desirable to combine administration of the anti-Ovr232v3 antibody or antibodies, with administration of an antibody directed against another tumor antigen associated with the particular cancer. As such, this invention is also directed to an antibody "cocktail" comprising one or more antibodies of this invention and at least one other antibody which binds another tumor antigen associated with the Ovr232v3- expressing tumor cells. The cocktail may also comprise antibodies that are directed to other epitopes of Ovr232v3. Preferably the other antibodies do not interfere with the binding and or internalization of the antibodies of this invention.

The antibody therapeutic treatment method of the present invention may involve the combined administration of an anti-Ovr232v3 antibody (or antibodies) and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include, e.g., estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).

The antibody may be combined with an anti-hormonal compound; e.g., an anti- estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen independent, the anti-Ovr232v3 antibody (and optionally other agents as described herein) may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy, before, simultaneously with, or post antibody therapy. Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-Ovr232v3 antibody. For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody is administered by intravenous infusion or.by subcutaneous injections. Depending on the type and severity of the 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 candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-Ovr232v3 antibody. However, other dosage regimens may be useful. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of a nucleic acid molecule encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody". See, for example, WO 96/07321 published March 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.

There are two major approaches to introducing the nucleic acid molecule (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery the nucleic acid molecule is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid molecule is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g. U.S. Patent Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acid molecules into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid molecule transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno- associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example). For review of the currently known gene marking and gene therapy protocols see Anderson et at., Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein.

Articles of Manufacture and Kits The invention also relates to an article of manufacture containing materials useful for the detection for Ovr232v3 overexpressing cells and/or the treatment of Ovr232v3 expressing cancer, in particular ovarian, colon, prostate, or lung cancer. The article of manufacture comprises a container and a composition contained therein comprising an antibody of this invention. The composition may further comprise a carrier. The article of manufacture may also comprise a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for detecting Ovr232v3 expressing cells and/or treating a cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti- Ovr232v3 antibody of the invention. The label or package insert indicates that the composition is used for detecting Ovr232v3 expressing cells and/or for treating ovarian, colon, prostate, or lung cancer, in a patient in need thereof. The label or package insert may further comprise instructions for administering the antibody composition to a cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a substance which detects the antibody of this invention, e.g., a second antibody which binds to the antibodies of this invention. The substance may be labeled with a detectable label such as those disclosed herein. The second container 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 materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Kits are also provided that are usefulfor various purposes , e.g., for Ovr232v3 cell killing assays, for purification or immunoprecipitation of Ovr232v3 from cells or for detecting the presence of Ovr232v3 in a serum sample or detecting the presence of Ovr232 v3 -expressing cells in a cell sample.. For isolation and purification of Ovr232v3, the kit can contain an anti-Ovr232v3 antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain the antibodies for detection and quantitation of Ovr232v3 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 therein comprising an antibody of this invention. The kit may further comprise a label or package insert on or associated with the container. The kits may comprise additional components, e.g., diluents and buffers, substances which bind to the antibodies of this invention, e.g., a second antibody which may comprise a label such as those disclosed herein, e.g., a radiolabel, fluorescent label, or enzyme, or the kit may also comprise control antibodies. The additional components may be within separate containers within the kit. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use. EXAMPLES

Example 1: Production and Isolation of Monoclonal Antibody Producing Hybridomas

The following MAb/hybridomas of the present invention are described below: Ovr232v3.Cl, Ovr232v3.C2, Ovr232v3.C3, Ovr232v3.C4, Ovr232v3.C5, Ovr232v3.C6, Ovr232v3.C7, Ovr232v3.C8, Ovr232v3.C9, Ovr232v3.C10, Ovr232v3.Cl l, Ovr232v3.C12, Ovr232v3.C13, Ovr232v3.C14, Ovr232v3.C15, Ovr232v3.C16, Ovr232v3.C17, Ovr232v3.C18, Ovr232v3.C19, Ovr232v3.C20, Ovr232v3.C21, Ovr232v3.C22, Ovr232v3.C23, Ovr232v3.C24, Ovr232v3.C25, Ovr232v3.C26, Ovr232v3.C27, Ovr232v3.C28, Ovr232v3.C29, Ovr232v3.C30, Ovr232v3.C31, Ovr232v3.C32, Ovr232v3.C33, Ovr232v3.C34, Ovr232v3.C35₃ Ovr232v3.C36, Ovr232v3.C37, Ovr232v3.C38, Ovr232v3.C39, Ovr232v3.C40, Ovr232v3.C41, Ovr232v3.C42, Ovr232v3.C43, Ovr232v3.C44, Ovr232v3.C45, Ovr232v3.C46, Ovr232v3.C47, Ovr232v3.C48, Ovr232v3.C49, Ovr232v3.C50, Ovr232v3.C51, Ovr232v3.C52, Ovr232v3.C53, Ovr232v3.C54, Ovr232v3.C55, Ovr232v3.C56, Ovr232v3.C57, Ovr232v3.C58, Ovr232v3.C59, Ovr232v3.C60, Ovr232v3.C61, Ovr232v3.C62, Ovr232v3.C63, Ovr232v3.C64, Ovr232v3.C65, Ovr232v3.C66, Ovr232v3.Dl, Ovr232v3.D2, Ovr232v3.D3, Ovr232v3.D4, Ovr232v3.D5, Ovr232v3.D6, Ovr232v3.D7, Ovr232v3.D8, Ovr232v3.D9, Ovr232v3.D10, Ovr232v3.Dl l, Ovr232v3.D12, Ovr232v3.D13, Ovr232v3.D14, Ovr232v3.D15, Ovr232v3.D16, Ovr232v3.D17, Ovr232v3.D18, Ovr232v3.D19, Ovr232v3.D20, Ovr232v3.D21, Ovr232v3.D22, Ovr232v3.D23, Ovr232v3.D24, Ovr232v3.D25, Ovr232v3.D26, Ovr232v3.D27, Ovr232v3.D28, Ovr232v3.D29, Ovr232v3.D30, Ovr232v3.D31, Ovr232v3.D32, Ovr232v3.D33, Ovr232v3.D34, Ovr232v3.D35, Ovr232v3.D36, Ovr232v3.D37, Ovr232v3.D38, Ovr232v3.D39, Ovr232v3.D40, Ovr232v3.D41,

Ovr232v3.D42, Ovr232v3.D43, and Ovr232v3.D44. If the MAb has been cloned, it will get the nomenclature "X.1," e.g., the first clone of Ovr232v3.C12 will be referred to as C12.1, the second clone of C12 will be referred to as C12.2, etc. For the purposes of this invention, a reference to Ovr232v3.C12 or C12 will include all clones, e.g., C12.1, C12.2, etc. Immunogens and Antigens (Recombinant Proteins, HA and His Tags and Transfected Cells)

For the Ovr232v3 Constructs described below, nucleic acid molecules encoding regions of Ovr232v3 were inserted into various expression vectors to produce recombinant proteins. These nucleic acid sequences were isolated using the primers included in the descriptions below of each construct.

For purposes of illustration, the predicted amino acid sequence encoded by each construct is also included. However, the constructs may include naturally occurring variants (e.g. allelic variants, SNPs) within the Ovr232v3 region as isolated by the primers. These variant sequences, and antibodies which bind to them are considered part of the invention as described herein.

Ovr232v3 Construct 1 Sequence and Protein Production

A nucleic acid molecule encoding the unique 76 amino acids in the extra cellular domain of Ovr232v3 protein sequence, Gly26 to LeulO2, was inserted into a modified pCMV5His3 vector at the EcoRI/Nhel sites. The nucleic acid molecule was isolated using the following primers:

- 5 '-pr-imer : CGGGATATCCATCACCATCACCATCACCATCACCATCACGGTGAGGCGCGGATTGGAGCAGAG (SEQ ID NO:1) 3'primer: CGGCTAGCTTATCAAAGGCGAGCAAAGCTGCGAAGTGAG (SEQ ID NO: 2)

The modified vector comprises a nucleotide sequence encoding a 17 amino acid secretion signal sequence from human stanniocalcin 1 (STCl) and a sequence encoding a 10 His tag. The resulting vector with the inserted Ovr232v3 nucleic acid fragment encodes a recombinant Ovr232v3 fusion protein with the STCl secretion signal fused to the N-terminus and the 10 His-tag fused to the C-terminus of the Ovr232v3 protein fragment (Gly26-Leul01). This recombinant plasmid encoding the Ovr232v3 unique region His-tagged protein is herein referred to as "Ovr232v3 Construct 1". A representative amino acid sequence encoded by Ovr232v3 Construct 1 is presented in SEQ ID NO:3.

Ovr232v3 Construct 1 Amino Acid Sequence (SEQ ID NO: 3)

1 11 21 31 41 51 I I I I I I

1 MLQNSAVLLV LVISASADIG EARIGAELWS WAGLGGSGPR PSAPETGIIG RGPRGRAFQR 61 GDRTVRPCSG SGPPRGRKRR GPSRGAASLR SFARLASHHH HHHHHHH The recombinant plasmid, Ovr232v3 Construct 1, was used to transfect HEK293F cells in suspension culture (1-10 liter serum free medium) in a spinner flask. Culture medium was harvested at 48 hours post-transfection. Medium was concentrated 10-100 fold, and diafiltrated with 100 mM sodium phosphate, 400 mM NaCl, 10% glycerol, pH 8.0. Concentrated medium containing protein encoded by Ovr232v3 Construct 1 was passed through a 5-mL nickel metal chelating column (His-Select-Ni, Sigma Inc., St. Louis, MO), which had been previously equilibrated with 100 mM sodium phosphate, 40OmM NaCl, 10% glycerol, pH 8.0. The column was then washed with 6 column volume (CV) of 100 mM sodium phosphate, 40OmM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0. Protein encoded by Ovr232v3 Construct 1 was eluted from the column using 22 CV of 100 mM sodium phosphate, 400 mM NaCl, 10% glycerol, pH 8.0 containing 50 or 500 mM imidazole, respectively. Samples from collected fractions were subjected to SDS- PAGE and Western blot analysis for assessing the purity of the protein. Purified fractions were pooled and dialyzed against PBS, pH 7.4. Ovr232v3 Construct 2 and Construct 3 Sequence and Protein Production

A nucleic acid molecule encoding the unique 76 amino acids in the extra cellular domain of Ovr232v3 protein sequence, Gly26 to LeulO2, fused with a STCl secretion signal at the N-terminus, was excised from Construct 1, described above, at the Pmel/Nhel sites and inserted into a modified human Fc fusion pTT3 vector at the Pmel/Nhel sites. The resulting vector encodes a recombinant Ovr232v3 fusion protein with the

STCl secretion signal fused to the N-terminus and the human Fc region fused to the C- terminus of the Ovr232v3 protein fragment (Gly26-Leul01). This recombinant plasmid encoding the Ovr232v3 unique region human Fc protein is herein referred to as "Ovr232v3 Construct 2". A representative amino acid sequence encoded by Ovr232v3 Construct 2 is presented in SEQ ID NO:4.

Ovr232v3 Construct 2 Amino Acid Sequence (SEQ ID NO: 4)

1 11 21 31 41 51 I I 1 I I I 1 MLQNSAVLLV LVISASADIG EARIGAELWS WAGLGGSGPR PSAPETGIIG RGPRGRAFQR

61 GDRTVRPCSG SGPPRGRKRR GPSRGAASLR SFARLASTCP PCPAPELLGG PSVFLFPPKP

121 KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQFN STFRVVSVLT

181 VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KTKGQPREPQ VYTLPPSREE MTKNQVSLTC

241 LVKGFYPSDI AVEWESSGQP ENNYNTTPPM LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV 301 MHEALHNHYT QKSLSLSPGK The same sequence encoding the unique 76 amino acids in the extra cellular domain of Ovr232v3 protein sequence, Gly26 to LeulO2, fused with a STCl secretion signal at the N-terminus, was excised out from the above modified human Fc fusion pTT3 expression vector (Ovr232v3 Construct 2) at the Pmel/Notl sites and inserted into another modified pTT3 vector.. The new vector contains a sequence encoding the Fc region of mouse immunoglobin G2.

The resulting vector encodes a recombinant Ovr232v3 fusion protein with the STCl secretion signal fused to the N-terminus and the mouse Fc region fused to the C- terminus of the Ovr232v3 protein fragment (Gly26-Leul01). This recombinant plasmid encoding the Ovr232v3 unique region mouse Fc protein is herein referred to as "Ovr232v3 Construct 3". A representative amino acid sequence encoded by Ovr232v3 Construct 3 is presented in SEQ ID NO:5.

Ovr232v3 Construct 3 Amino Acid Sequence (SEQ ID NO: 5) 1 11 21 31 41 51

I I I I I I

1 MLQNSAVLLV LVISASADIG EARIGAELWS WAGLGGSGPR PSAPETGIIG RGPRGRAFQR

61 GDRTVRPCSG SGPPRGRKRR GPSRGAASLR SFARLASENL YFQGPRGPTI KPCPPCKCPA

121 PNLLGGPSVF IFPPKIKDVL MISLSPIVTC VVVDVSEDDP DVQISWFVNN VEVHTAQTQT 181 HREDYNSTLR VVSALPIQHQ DWMSGKEFKC KVNNKDLPAP IERTISKPKG SVRAPQVYVL

241 PPPEEEMTKK QVTLTCMVTD FMEEDIYVEW TNNGKTELNY- KNTEPVLDSD GSYFMYSKLR ^' 30Ϊ VEKKNWVERN SYSCSVVHEG LHNHHTTKSF SRTPGK

The recombinant plasmids, Ovr232v3 Construct 2 and Ovr232v3 Construct 3, were used to independently transfect HEK293F cells (Invitrogen, Carlsbad, CA) in suspension culture (1 liter serum free medium) in spinner flasks. Culture medium was harvested at 48 hours post-transfection. Medium was concentrated about 10-fold, diafiltrated into 2X PBS with 50 mM sodium phosphate, pH 8.0, and filtered through a 0.45 μM membrane. Concentrated medium containing Ovr232v3 Fc fusion proteins encoded by either Construct 2 or Construct 3 was passed over a 10-mL protein A affinity column (Protein A Fast Flow Sepharose, Amersham Biosciences, Piscataway, NJ), which had been previously equilibrated with 5OmM sodium borate and 2X PBS, pH 8.0 (Buffer A). The column was then washed with 6 column volume (CV) of Buffer A. Ovr232v3 Fc fusion protein was eluted from the column by switching to Buffer B (0.1 M sodium citrate, pH 3.0). Each fraction was neutralized with ¹A volume of 1 M Tris/HCl, pH 8.0. Samples from collected fractions were subjected to SDS-PAGE and Western blot analysis for assessing the purity of Ovr232v3 Fc fusion proteins encoded by Construct 2 and Construct 3. Purified fractions were pooled and dialyzed against 2X PBS, pH 7.4.

Ovr232v3 Construct 4 Sequence and Protein Production

A nucleic acid molecule encoding the full length Ovr232v3 (Metl-Ala390) was cloned into a mammalian expression vector, pCMV5His3, without any tags at the

Pmel/Nhel sites to generate an Ovr232v3 expressing vector. The nucleic acid molecule was isolated using the following primers:

5' primer: CTTTGTTTAAACATGGCGCCCCCGCAGGTCCTC (SEQ ID NO: 6) 3' primer: CGGCTAGCTTATCATGCATTGAGTTCCCTATGCATC (SEQ ID NO: 7)

The resulting vector encodes a full length Ovr232v3 protein with the native secretion signal, transmembrane domain, and cytoplasmic tail (Metl-Ala390). This recombinant plasmid encoding the full length Ovr232v3 protein is herein refered to as "Ovr232v3 Construct 4". A representative amino acid sequence encoded by Ovr232v3 Construct 4 is presented in SEQ ID NO:δ.

Ovr232v3 Construct 4 Amino Acid Sequence (SEQ ID NO: 8)

1 11 21 31 41 51 I I I I I I 1 MAPPQVLAFG LLLAAATATF AAAQEGEARI GAELWSWAGL GGSGPRPSAP ETGIIGRGPR

61 GRAFQRGDRT VRPCSGSGPP RGRKRRGPSR GAASLRSFAR LECVCENYKL AVNCFVNNNR

121 QCQCTSVGAQ NTVICSKLAA KCLVMKAEMN GSKLGRRAKP EGALQNNDGL YDPDCDESGL

181 FKAKQCNGTS MCWCVNTAGV RRTDKDTEIT CSERVRTYWI IIELKHKARE KPYDSKSLRT

241 ALQKEITTRY QLDPKFITSI LYENNVITID LVQNSSQKTQ NDVDIADVAY YFEKDVKGES 301 LFHSKKMDLT VNGEQLDLDP GQTLIYYVDE KAPEFSMQGL KAGVIAVIVV VVIAVVAGIV

361 VLVISRKKRM AKYEKAEIKE MGEMHRELNA

The Ovr232v3 Construct 4 expression vector was used to transfect human HEK293F cells from Invitrogen. Briefly, 50 ml of 293F cells cultured in GIBCO freestyle medium (Invitrogen) at 10⁶ cells/ml for 24 hr were transfected with an Ovr232v3

Construct 4 expression vector using 293fectin transfection reagent (Invitrogen,), according to the manufacturer's protocol. GIBCO OPTI-MEM medium (Invitrogen) was used to dilute DNA, cells and transfectant. Cells were harvested for analysis 48 hr post transfection. Ovr232v3 Construct 5 Sequence and Protein Production

A nucleic acid molecule encoding the full length Ovr232v3 (Metl-Ala390) was inserted into a mammalian expression vector, pcDNA5/FRT/TO (Invitrogen), at the Pmel/Nhel sites to generate an Ovr232v3 expressing vector. The nucleic acid molecule was isolated using the primers described above in Ovr232v3 Construct 4 (SEQ ID NO: 6 and SEQ ID NO: 7).

The resulting vector encodes a full length Ovr232v3 protein with the native secretion signal, transmembrane domain, cytoplasmic tail (Metl-Ala390) and a 10-His tag fused at the C-terminus. This recombinant plasmid encoding the His-tagged foil length Ovr232v3 protein is herein refered to as "Ovr232v3 Construct 5". A representative amino acid sequence encoded by Ovr232v3 Construct 5 is presented in SEQ ID NO:9.

Ovr232v3 Construct 5 Amino Acid Sequence (SEQ ID NO: 9) 1 11 21 31 41 51 I I I I I I i MAPPQVLAFG LLLAAATATF AAΆQEGEARI GAELWSWAGL GGSGPRPSAP ETGIIGRGPR 61 GRAFQRGDRT VRPCSGSGPP RGRKRRGPSR GAΆSLRSFAR LECVCENYKL AVNCFVNNNR

121 QCQCTSVGAQ NTVICSKLAA KCLVMKAEMN GSKLGRRAKP EGALQNNDGL YDPDCDESGL 181 FKAKQCNGTS MCWCVNTAGV RRTDKDTEIT CSERVRTYWI IIELKHKARE KPYDSKSLRT 241 ALQKEITTRY QLDPKFITSI LYENNVITID LVQNSSQKTQ NDVDIADVAY YFEKDVKGES 301 LFHSKKMDLT VNGEQLDLDP GQTLIYYVDE KAPEFSMQGL KAGVIAVIVV VVIAVVAGIV 361 VLVISRKKRM AKYEKAEIKE MGEMHRELNA ASHHHHHHHH HH CHO-FIpIn stable cell lines were prepared by transfection of CHO cells

(Invitrogen), (catalogue number: R758-07) with the pFRT/lacZeo vector (following Invitrogen's recommendation). CHO-FIpIn cells were cultured in HAM Fl 2 medium supplemented with 10% fetal bovine serum (FBS). The Ovr232v3 expressing stable cell line was generated by further co-transfection of the CHO-FIpIn cells with Ovr232v3 Construct 5 and pOG44 vector expressing FIp recombinase. Stable transfectants were selected in HamF12 medium +10%FBS with Hygromycin B at 300 ug/ml, for 15 days. Hydromycin B- resistant cells were checked for expression of Ovr232v3 Construct 5 protein by Western Blot using anti-EpCAM monoclonal antibody clone C-IO from Santa Cruz Biotech (Santa Cruz, CA), (catalogue number: sc-25308). The cells were further cloned, expanded, scaled-up, cryopreserved in FBS with 10% DMSO and stored in liquid nitrogen at -196 °C to assure maintenance of viable clone cultures.

Ovr232v3 Construct 6 Sequence and Protein Production

A nucleic acid molecule encoding a fragment of Ovr232v3 from Gln24 to Ala342 was cloned into a modified mammalian expression vector, pCMV5His3. The nucleic acid molecule was isolated using the following primers:

5' primer: CGGGATATCCAGGAAGGTGAGGCGCGGATTG (SEQ ID NO: 10) 3' primer: ATTCTCAATGCAGGGTCTAAAAGCTAGCCG (SEQ ID NO: 11) The modified vector comprises a nucleotide sequence encoding a 17 amino acid secretion signal sequence from human stanniocalcin 1 (STCl) and a sequence encoding a 10 His tag. The resulting vector with the inserted Ovr232v3 nucleic acid fragment encodes a recombinant Ovr232v3 fusion protein with the STCl secretion signal fused to the N-terminus and the 10 His-tag fused to the C-terminus of the Ovr232v3 protein fragment (Gln24-Ala342). This recombinant plasmid encoding the His-tagged Ovr232v3 protein fragment is herein refered to as "Ovr232v3 Construct 6". A representative amino acid sequence encoded by Ovr232v3 Construct 6 is presented in SEQ ID NO: 12.

Ovr232v3 Construct 6 Amino Acid Sequence (SEQ ID NO: 12)

1 11 21 31 41 51

I I I I I I

1 MLQNSAVLLV LVISASADIQ EGEARIGAEL WSWAGLGGSG PRPSAPETGI IGRGPRGRAF

61 QRGDRTVRPC SGSGPPRGRK RRGPSRGAAS LRSFARLECV CENYKLAVNC FVNNNRQCQC 121 TSVGAQNTVI CSKLAAKCLV MKAEMNGSKL GRRAKPEGAL QNNDGLYDPD CDESGLFKAK

181 QCNGTSMCWC VNTAGVRRTD KDTEITCSER VRTYWIIIEL KHKAREKPYD SKSLRTALQK

241 EITTRYQLDP KFITSILYEN NVITIDLVQN SSQKTQNDVD IADVAYYFEK DVKGESLFHS

301 KKMDLTVNGE QLDLDPGQTL IYYVDEKAPE FSMQGLKΆSH HHHHHHHHH The Ovr232v3 Construct 6 expression vector was used to transfect human

HEK293E cells from the Biotechnology Research Institute, National Research Council Canada (Quebec, Canada). HEK2936E cells (7 x 10⁵ cells/ml) were grown in Freestyle medium (Invitrogen) one day before the transfection (Alternatively, 1.5 x 10⁵ cells/ml of HEK293E cells were grown 3 days before transfection). The cell density was diluted to about 1.0-1.1 x 10⁶ cells/ml at the time of transfection. One mg of DNA was used to transfect one liter of the HEK293E cells at the above density. The final volume of the transfection reagents should be 1/10^th of the total cell culture volume. PEI-DNA complex was prepared in the Freestyle medium, at room temperature, in the ratio of 2:1 (2μg of PELlμg of DNA). After addition of PEI (Polyethyleninine linear MW 25,000, Ref # 23966 from Polysciences, Inc., Warrington, PA) (solution in Freestyle medium) to the DNA solution, the mixture was immediately vortexed and incubated for 15 minutes at room temperature and then added to the cell culture. Cells were harvested for analysis 48 hr post transfection.

Additionally, a fragment from the Ovr232v3 Construct 6 expression vector was excised out by cutting with Pmel and Notl restriction enzymes and inserted into the ρTT3 expression vector (NRC Canada). This derivative of the Ovr232v3 Construct 6 expression vector was used to produce protein for use in the examples below. Ovr232v3 Construct 7 Sequence and Protein Production

A nucleic acid molecule encoding the extra cellular domain of Ovr232v3 (Metl- Ala342) was cloned into a modified mammalian expression vector, ρTT3hFc, at the Pmel/Nhel sites to generate an Ovr232v3 expressing vector. The nucleic acid molecule was isolated using the following primers:

5' primer: CTTTGTTTAAACATGGCGCCCCCGCAGGTCCTC (SEQ ID NO: 13) 3' primer: CGGCTAGCTGCATTGAGTTCCCTATGCATC (SEQ ID NO: 14)

The resulting vector encodes a recombinant Ovr232v3 fusion protein with the Ovr232v3 extracellular domain, including the native secretion sequence (Metl to Lys341), and the human Fc domain fused to the C-terminus. This recombinant plasmid encoding the Ovr232v3 extracellular domain fused to human Fc is herein refered to as "Ovr232v3 Construct 7". A representative amino acid sequence encoded by Ovr232v3 Construct 6 is presented in SEQ ID NO: 15.

Ovr232v3 Construct 7 Amino Acid Sequence (SEQ ID NO: 15)

1 11 21 31 41 51

I I I I I I 1 MAPPQVLAFG LLLAAATATF. AAAQEGEARI GAELWSWAGL GGSGPRPSAP ^"ETGIIGRGPR

^"6Ϊ GRAFQRGDRT VRPCSGSGPP RGRKRRGPSR GAASLRSFAR LECVCENYKL AVNCFVNNNR

121 QCQCTSVGAQ NTVICSKLAA KCLVMKAEMN GSKLGRRAKP EGALQNNDGL YDPDCDESGL

181 FKAKQCNGTS MCWCVNTAGV RRTDKDTEIT CSERVRTYWI IIELKHKARE KPYDSKSLRT

241 ALQKEITTRY QLDPKFITSI LYENNVITID LVQNSSQKTQ NDVDIADVAY YFEKDVKGES 301 LFHSKKMDLT VNGEQLDLDP GQTLIYYVDE KAPEFSMQGL KAASTCPPCP APELLGGPSV

361 FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQFNSTF

421 RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT LPPSREEMTK

581 NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL TVDKSRWQQG

641 NVFSCSVMHE ALHNHYTQKS LSLSPGK

The Ovr232v3 Construct 7 expression vector was used to produce protein for use in the examples below.

Ovr232v3 Construct 8 Sequence and Protein Production

A nucleic acid molecule encoding the full length Ovr232v3 (Metl-Ala390) was cloned into a mammalian expression vector, PCMV5His2, at the Pmel/Xbal sites to generate an Ovr232v3 expressing vector with c-terminal HA-tag. The nucleic acid molecule was isolated using the following primers:

5' primer: AGCTTTGTTTAAACATGGCGCCCCCGCAG (SEQ ID NO: 16)

3^r primer: CGGCTAGCTTATCATGCATTGAGTTCCCTATGCATC (SEQ ID NO: 16) The resulting vector encodes a full length Ovr232v3 protein with the native secretion signal, transmembrane domain, cytoplasmic tail (Metl-Ala390), and a HA-tag fused to the C-terminus. This recombinant plasmid encoding the HA-tagged full length Ovr232v3 protein is herein refered to as "Ovr232v3 Construct 8". A representative amino acid sequence encoded by Ovr232v3 Construct 8 is presented in SEQ ID NO: 18.

Ovr232v3 Construct 8 Amino Acid Sequence (SEQ ID NO: 18)

1 11 21 31 41 51

I I I 1 I I 1 MAPPQVLAFG LLLAAATATF AAAQEGEARI GAELWSWAGL GGSGPRPSAP ETGIIGRGPR

61 GRAFQRGDRT VRPCSGSGPP RGRKRRGPSR GAASLRSFAR LECVCENYKL AVNCFVNNNR

121 QCQCTSVGAQ NTVICSKLAA KCLVMKAEMN GSKLGRRAKP EGALQNNDGL YDPDCDESGL

181 FKAKQCNGTS MCWCVNTAGV RRTDKDTEIT CSERVRTYWI IIELKHKARE KPYDSKSLRT

241 ALQKEITTRY QLDPKFITSI LYENNVITID LVQNSSQKTQ NDVDIADVAY YFEKDVKGES 301 LFHSKKMDLT VNGEQLDLDP GQTLIYYVDE KAPEFSMQGL KAGVIAVIVV VVIAVVAGIV

361 VLVISRKKRM AKYEKAEIKE MGEMHRELNA GYPYDVPDYA

The Ovr232v3 Construct 8 recombinant plasmid was used to transiently transfect 293T cells (ATCC, Manassus, VA). Ovr232v3 Construct 9 Sequence and Protein Production

A nucleic acid molecule encoding the full length Ovr232v3 (Metl-Ala390) was inserted into retroviral vector PLXSN (BD Biosciences Clontech, Palp Alto, CA), at the - Hpal site to generate an Ovr232v3 retroviral vector. The nucleic acid molecule was isolated using the primers described above in Ovr232v3 Construct 4 (SEQ ID NO: 6 and SEQ ID NO: 7).

The resulting vector encodes a full length Ovr232v3 protein with the native secretion signal, transmembrane domain, cytoplasmic tail (Metl-Ala390). This recombinant plasmid encoding the retroviral full length Ovr232v3 protein is herein refered to as "Ovr232v3 Construct 9". A representative amino acid sequence encoded by Ovr232v3 Construct 9 is presented in SEQ ID NO: 19.

Ovr232v3 Construct 9 Amino Acid Sequence (SEQ ID NO: 19)

1 11 21 31 41 51 I I 1 I I I 1 MAPPQVLAFG LLLAAATATF AAAQEGEARI GAELWSWAGL GGSGPRPSAP ETGIIGRGPR

61 GRAFQRGDRT VRPCSGSGPP RGRKRRGPSR GAASLRSFAR LECVCENYKL AVNCFVNNNR

121 QCQCTSVGAQ NTVICSKLAA KCLVMKAEMN GSKLGRRAKP EGALQNNDGL YDPDCDESGL

181 FKAKQCNGTS TCWCVNTAGV RRTDKDTEIT CSERVRTYWI IIELKHKARE KPYDSKSLRT

241 ALQKEITTRY QLDPKFITSI LYENNVITID LVQNSSQKTQ NDVDIADVAY YFEKDVKGES 301 LFHSKKMDLT VNGEQLDLDP GQTLIYYVDE KAPEFSMQGL KAGVIAVIVV VVIAVVAGIV

361 VLVISRKKRM AKYEKAEIKE MGEMHRELNA The Ovr232v3 Construct 9 was used to produce retrovirus for RK3E cell (ATCC, Manassus, VA) infection.

Ovr232v3 Construct 10 Sequence and Protein Production

A nucleic acid molecule encoding the full length Ovr232v3 (Metl-Ala390) was inserted into retroviral vector PLPCX (BD Biosciences Clontech, Palo Alto, CA) between HindIII and Notl sites to generate an Ovr232v3 retroviral vector. The nucleic acid molecule was isolated using the primers described above in Ovr232v3 Construct 4 (SEQ ID NO: 6 and SEQ ID NO: 7).

The resulting vector encodes a full length Ovr232v3 protein with the native secretion signal, transmembrane domain, cytoplasmic tail (Metl-Ala390). This recombinant plasmid encoding the retroviral full length Ovr232v3 protein is herein refered to as "Ovr232v3 Construct 10". A representative amino acid sequence encoded by Ovr232v3 Construct 10 is presented in SEQ ID NO: 20.

Ovr232v3 Construct 10 Amino Acid Sequence (SEQ ID NO: 20)

1 11 21 31 41 51 I I I I I I

1 MAPPQVLAFG LLLAAATATF AAAQEGEARI GAELWSWAGL GGSGPRPSAP ETGIIGRGPR

61 GRAFQRGDRT VRPCSGSGPP RGRKRRGPSR GAASLRSFAR LECVCENYKL-AVNCFVNNNR ■ -121- QCQCTSVGAQ NTVICSKLAA KCLVMKAEMN GSKLGRRAKP EGALQNNDGL YDPDCDESGL

181 FKAKQCNGTS TCWCVNTAGV RRTDKDTEIT CSERVRTYWI IIELKHKARE KPYDSKSLRT 241 ALQKEITTRY QLDPKFITSI LYENNVITID LVQNSSQKTQ NDVDIADVAY YFEKDVKGES 301 LFHSKKMDLT VNGEQLDLDP GQTLIYYVDE KAPEFSMQGL KAGVIAVIVV VVIAVVAGIV 361 VLVISRKKRM AKYEKAEIKE MGEMHRELNA

The Ovr232v3 Construct 10 was used to produce retrovirus for HCTl 16 cell

(ATCC, Manassus, VA) infection to generate HCTl 16-Ovr232v3 cells.

Ep-CAM Construct 1 Sequence and Protein Production

A nucleic acid molecule encoding the full length EpCAM (Metl-Ala314) was inserted into a mammalian expression vector, PCMV5His2, at the Pmel/Xbal sites to generate an Ep-CAM expressing vector with c-terminal HA-tag. The nucleic acid molecule was isolated using the following primers:

5' primer: AGCTTTGTTTAAACATGGCGCCCCCGCAG (SEQ ID NO: 21) 3' primer: CTAGTCTAGATTATCACGCGTAGTCCGGCACGTCGTACGG (SEQ ID NO: 22)

The resulting vector encodes a full length Ep-CAM protein with the native secretion signal, transmembrane domain, cytoplasmic tail (Metl-Ala314) and a HA-tag fused at the C-terminus. This recombinant plasmid encoding the HA-tagged full length Ep-CAM protein is herein refered to as "Ep-CAM Construct 1". A representative amino acid sequence encoded by Ep-CAM Construct 1 is presented in SEQ ID NO: 23.

Ep-CAM Construct 1 Amino Acid Sequence (SEQ ID NO: 23) 1 11 21 31 41 51

I I I I I I

1 MAPPQVLAFG LLLAAATATF AAAQEECVCE NYKLAVNCFV NNNRQCQCTS VGAQNTVICS 61 KLAAKCLVMK AEMNGSKLGR RAKPEGALQN NDGLYDPDCD ESGLFKAKQC NGTSTCWCVN 121 TAGVRRTDKD TEITCSERVR TYWIIIELKH KAREKPYDSK SLRTALQKEI TTRYQLDPKF 181 ITSILYENNV ITIDLVQNSS QKTQNDVDIA DVAYYFEKDV KGESLFHSKK MDLTVNGEQL 241 DLDPGQTLIY YVDEKAPEFS MQGLKAGVIA VIVVVVIAVV AGIVVLVISR KKRMAKYEKA 301 EIKEMGEMHR ELNAGYPYDV PDYA

The Ep-CAM Construct 1 was used to transiently transfect 293 T cells. Ep-CAM Construct 2 Sequence and Protein Production

A nucleic acid molecule encoding the full length EpCAM (Metl-Ala314) was inserted into retroviral vector PLXSN (BD Biosciences Clontech ), at the Hpal site to generate an Ovr232v3 retroviral vector. The nucleic acid molecule was isolated using the primers described above in Ep-CAM Construct 1 (SEQ ID NO: 21 and SEQ ID NO: 22). The resulting vector encodes a full length Ep-CAM protein with the native secretion signal, transmembrane domain, and cytoplasmic tail (Metl-Ala314). This recombinant plasmid encoding the retroviral full length Ep-CAM protein is herein refered to as "Ep-CAM Construct 2". A representative amino acid sequence encoded by Ep-CAM

Construct 2 is presented in SEQ ID NO: 24.

Ep-CAM Construct 2 Amino Acid Sequence (SEQ ID NO: 24)

1 11 21 31 41 51 I I I I I I

1 MAPPQVLAFG LLLAAATATF AAAQEECVCE NYKLAVNCFV NNNRQCQCTS VGAQNTVICS 61 KLAAKCLVMK AEMNGSKLGR RAKPEGALQN NDGLYDPDCD ESGLFKAKQC NGTSTCWCVN 121 TAGVRRTDKD TEITCSERVR TYWIIIELKH KAREKPYDSK SLRTALQKEI TTRYQLDPKF 181 ITSILYENNV ITIDLVQNSS QKTQNDVDIA DVAYYFEKDV KGESLFHSKK MDLTVNGEQL 241 DLDPGQTLIY YVDEKAPEFS MQGLKAGVIA VIVVVVIAW AGIVVLVISR KKRMAKYEKA 301 EIKEMGEMHR ELNA

The Ep-CAM Construct 2 was used to produce retrovirus for RK3E cell (ATCC) infection.

Generation of stably-infected RK3 E cell lines

RK3E cells stably expressing Ovr232v3 or Ep-CAM and control gene alkaline phosphatase (AP) were generated using the Phoenix Retrovirus Expression System (Orbigen, San Diego, CA). Ovr232v3 Construct 9, Ep-CAM Construct 2, and an AP encoding sequence inserted into retroviral vector PLXSN (pLAPSN, BD Biosciences Clontech, Palo Alto, CA), were transfected into Phoenix-Eco packaging cells. Two days later, the culture media were harvested and filtered through 0.45um polysulfonic filter. RK3E cells were split at 5x10⁵ cells on 10 cm plates the day before the infection. 8ug/ml polybrene (Sigma- Aldrich, St. Louis, MO) was added to the virus-containing media prior to their addition to the target cells. 7 hours later, the virus media was replaced with fresh growth medium. Stably-infected cells were selected with 0.5 mg/ml G418.

RK3E cells stably expressing Ovr232v3, Ep-CAM or AP are refered to herein as RK3E-Ovr232v3, RK3E-EpCAM₅ and RK3E-AP, respectively.

Immunizations In order to generate anti-Ovr232v3 MAbs with utility as in- vivo therapeutic agents, and in-vivo and in-vitro diagnostic agents, mice were immunized with various Ovr232v3 constructs.

For the C-series MAb fusion, mice were immunized with the protein encoded by

Ovr232v3 Construct 3 (described above). The protein was expressed in mammalian cells. For the D-series MAb fusion, mice were immunized with the protein encoded by

Ovr232v3 Construct 6 (described above). The protein was expressed in mammalian cells. . For both the C- and D-series immunizations, groups of 8 BALB/c mice were immunized intradermally in both rear footpads. All injections were 25 uL per foot. The first injection of 10 ug of antigen per mouse was in Dulbecco's phosphate buffered saline (DPBS) mixed in equal volume to volume ratio with Titermax gold adjuvant (Sigma).

Subsequently, mice were immunized twice weekly for 5 weeks. For the 2^nd through 9^th injection, mice were immunized with 10 ug of antigen in 20 uL of DPBS plus 5uL of

Adju-phos adjuvant (Accurate Chemical & Scientific Corp., Westbury, NY) per mouse.

The final immunization consisted of 10 ug antigen diluted in DPBS alone. Hybridoma Fusion

Four days after the final immunization, mice were sacrificed and draining lymph node (popliteal) tissue was collected by sterile dissection. Lymph node cells were dispersed using a Tenbroeck tissue grinder (Wheaton #347426, VWR, Brisbane, CA) followed by pressing through a sterile sieve (VWR) into DMEM and removing T-cells via anti-CD90 (Thy 1.2) coated magnetic beads (Miltenyi Biotech, Bergisch-Gladbach, Germany). These primary B-cell enriched lymph node cells were then immortalized by electro-cell fusion (BTX, San Diego, CA) with the continuous myeloma cell line P3x63Ag8.653 (Kearney, J.F. et al, J. Immunology 123: 1548-1550, 1979). The myeloma and B-cells were pooled at a 1 : 1 ratio for the fusion. These fusion cultures were distributed at 2 million cells per plate into wells of 96 well culture plates (Costar #3585, VWR). Successfully fused cells were selected by culturing in selection medium (DMEM/15% FBS) containing 2.85 μM Azaserine, 50 μM Hypoxanthine (HA) (Sigma) or 50 μM Hypoxanthine, 0.2 μM Aminopterin, 8 μM Thymidine (HAT) (Sigma) supplemented with recombinant human IL-6 (Sigma) at 0.5ng/mL. Cultures were transitioned into medium (DMEM/10% FBS) without selection and IL-6 supplements for continued expansion and antibody production.

Supernatants from wells were screened by enzyme linked solid phase immunoassay (ELISA), flow cytometry and antibody internalization for reactivity against Ovr232v3. Monoclonal cultures, consisting of the genetically uniform progeny from 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, FL). The resulting murine B-cell hybridoma cultures were expanded using standard tissue culture techniques. Selected hybridomas were cryopreserved in fetal bovine serum (FBS) with 10% DMSO and stored in Liquid Nitrogen at -196⁰C to assure maintenance of viable clone cultures.

ELISA Screening and Selection of Hybridomas Producing Oyr232v3 Specific Antibodies - C Series

Hybridoma cell lines were selected for production of Ovr232v3 specific antibody by ELISA. Four different assay formats were used. ELISA Screen Format 1

In format 1 (direct ELISA), the protein encoded by Ovr232v3 Construct 2 (described above) was incubated overnight at 1 ug/mL in PBS, lOOuL / well in 96 well polystyrene EIA plates (Costar #9018, VWR) at 4⁰C. The plate wells were washed twice with Tris buffered saline with 0.05% Tween20, pH 7.4 (TBST). Nonspecific binding capacity was blocked by filling the wells (300ul/well) with TBST/0.5% bovine serum albumin (TBST/BSA) and incubating for >30 minutes at room temperature (RT). ELISA Screen Format 2

In format 2 (His Capture Sandwich ELISA), 100 ul antigen solution per well was added to High Binding HisGrab plates (Pierce P/N 15143) and incubated overnight at 4⁰C. The antigen solution was conditioned medium of the protein encoded by Ovr232v3 Construct 4 (described above) diluted 1 :20 with TBST/BSA. The plate wells were washed and blocked as described in format 1.

ELISA Screen Format 3 and 4

In formats 3 and 4 (human Fc Capture Sandwich ELISA), goat anti-human IgG Fc with minimal cross-reactivity to mouse Fc (2 ug/mL in PBS; 100 uL / well; Jackson Immunoresearch P/N 109-005-098, West Grove, PA) was nonspecifically adsorbed to the surface of the EIA plates by incubating overnight at 4⁰C. The plate wells were emptied and nonspecific binding capacity was blocked as described in format 1. Then, antigen solution was added. For format 3 the antigen solution was the purified protein encoded by Ovr232v3 Construct 2 (described above) at lug/mL in TBST/BSA. For format 4 the antigen solution was conditioned medium, diluted 1 :20 in

TBST/BSA, of 293F cells transfected with Ovr232v3 Construct 7. Incubation with the antigen solution was for >1 hr at room temperature.

Further processing of plates was identical for all 4 formats. The wells were emptied and filled with 5OuL / well TBST/BSA to prevent them from drying out during the sample collection process. Hybridoma culture medium sample was added to the wells (5OuL) and incubated for 1 hour at RT. The wells were washed 3 times with TBST. One hundred uL of alkaline phosphatase conjugated goat anti-mouse IgG (Fc) with minimal cross-reactivity to human Fc (P/Nl 15-055-071, Jackson Immunoresearch), diluted 1:5000 in TBST/BSA, was added to each well and incubated for >1 hour at RT. The wells were washed 3 times with TBST. One hundred uL of alkaline phosphatase substrate para- nitrophenylphosphate (pNPP) (Sigma) at lmg/mL in 1 M Diethanolamine buffer pH 8.9 (Pierce) was added to each well and incubated for 20 min at RT. The enzymatic reaction was quantified by measuring the solution's absorbance at 405 run wavelength.

The data from all four screening assay formats were evaluated and a cut-off for selection of 0.75 OD was established for each assay. Cultures with supernatants which produced absorbance values greater than 0.75 in any one of the four assay formats were expanded for further analysis. Antibodies from selected hybridomas were evaluated for cross-reactivity to human IgG Fc by a direct ELISA assay with three human IgG MAbs as coated antigen, and in a sandwich ELISA assay with human Fc domain fused to a nonrelevant protein as the antigen preparation. Ovr232v3.C62 and Ovr232v3.C65 showed cross-reactivity in both of these assays. All other monoclonal antibodies were specific to Ovr232v3 in these assays.

Flow Cytometry Screening for Cell Surface Binding of Oyr232v3 MAbs — C Series

293 F cells were transiently transfected with the Ovr232v3 Construct 4 expression plasmid (described above) using 293fectin (Invitrogen) as transfection reagent. 48 hours post-transfection, cells were washed once with 10ml Ca⁺²/Mg⁺² free DPBS and then 7ml of warm (37°C) Cellstripper (Mediatech, Herndon, VA) was added per 150cm flask. The cells were then incubated for 5 minutes at 37⁰C with tapping of the flask to remove tightly attached cells. The cells were removed and pipetted several times to break aggregates, then immediately placed in DMEM/10% FBS/5mM sodium butyrate. The cells were then centrifuged down for 5 minutes at 1300 rpm and resuspended in DMEM/10% FBS/5mM sodium butyrate. The cells were incubated at 37⁰C for a 30 min. recovery period. Prior to staining, viability of the cells was measured using Guava Viacount (Guava Cytometers, Foster City, CA) and cultures with > 90% viability were selected for staining with MAbs. Cells from cultures selected for MAb staining were aliquoted at 0.5 -1.0x10⁶ cells/well in 96-well v-bottom plates (VWR) and centrifuged for 2 minutes at 1500 rpm. Supernatants were aspirated and plates briefly shaken on a vortex mixer to resuspend the 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 or purified MAb was added to the cells. Plates were stored on ice for 15 min., then washed and centrifuged as above, in 200 uL of FACS buffer. This washing procedure was repeated twice and then 25 uL of phycoerythrin (PE) conjugated donkey anti-mouse IgG Fc antibody (Jackson Immunoresearch Laboratories) were added to cells. After 15 minutes on ice the cells were washed twice, as above and then resuspended in 250 uL of FACS buffer for analysis on the cell sorter or flow cytometer. In certain cases, for storage overnight at 4⁰C prior to analysis, 133 ul of FACS buffer and 67 uL of 1% paraformaldehyde/DPBS was added to each well, for fixation, then the volume was increased to 250 uL with DPBS. Stained cells were analyzed on an Elite fluorescent activated cell sorter (FACS) (Beckman-Coulter).

Table 2. Cell surface staining of Ovr232v3 transfected 293 F cells with supernatants of uncloned h bridomas.

Cloning of Hybridomas Producing Oyr232v3 Specific MAb - C Series

Based on data from ELISA, flow cytometry, and internalization experiments (see

Example 3 below), the following hybridomas were selected for single cell cloning into 96 well culture plates by cell sorting (Coulter Elite): C8, C9, C12, C14, C15, C16, C18, C20,

C26, C29, C30, C31, C32, C59, and C64.

After 2 weeks of culture, supernatants from up to 3 hybridoma clones from each parent hybridoma were tested for staining of Ovr232v3 transfected 293F cells by flow cytometry. 293F cells transfected with a construct encoding Ovrl 10 (also known as B7-

H4) were used as negative control. Antibodies were purified from supernatants of selected clones and re-analyzed by staining of Ovr232v3-transfected 293F cells and tumor cells. The following tumor cell lines were used: SW620, HCTl 16 and HeLa. SW620 and HCTl 16 express Ovr232v3 RNA as determined by QPCR. HeLa cells do not express Ovr232v3 RNA. Ricin does not localized to the cell surface and was used as a negative control. CD71 does localized at the cell surface and was used as a positive control. Results are shown in tables 3 and 4 below.

Table 3. Cell surface stainin of Oyr232v3 transfected 293F cells with urified MAbs.

I C64.2 J 8.4 I 0,912 | 14.3 | 1.07 [ 7.9 | 1.17 I

All selected antibodies shown in Table 3 recognize native Ovr232v3 protein on the plasma membrane of transfected 293F cells. Anti-Ovr232v3 MAbs bind to tumor cells expressing Ovr232v3 as shown in Table 4 above. Antibodies C9.1, C16.1, C18.1, C20.1, C26.1, C30.1 and C64.2 all demonstrate strong binding to Ovr232v3 on tumor cells and antibodies C9.1, C18.1, C30.1 and C64.2 did demonstrate cell surface binding to HeLa cells, indicating high specificity for Ovr232v3.

ELISA Screening and Selection of Hybridomas Producing Oyr232v3 Specific Antibodies - D Series Hybridoma cell lines were selected for production of Ovr232v3 recognizing antibody by direct ELISA, Sandwich ELISA and Cell ELISA. The protein encoded by Ovr232v3 Construct 6 was used as antigen for the direct ELISA. The Sandwich ELISA followed format 4 as described for the screening of C series hybridomas. Ovr232v3 Construct 7 and Construct 2 were used as antigens. For the Cell ELISA, the binding of antibodies to either stably transduced RK3E-Ovr232v3 cells or RK3E-AP cells was evaluated. 25,000 cells in 100 ul growth medium were plated.p.er well of a 96-well plate coated with Poly-D-Lysine (#15600, Pierce). Cells were incubated overnight, and 50 ul hybridoma supernatant or purified antibody (1 ug/ml final concentration) were added to each well. Cells were incubated on ice for 30 min. Wells were emptied and washed with TBST/BS A. Cells were then fixed for 10 min on ice by adding 100 ul 4% formaldehyde in TBS. Wells were emptied and washed with TBST/BSA. 300 ul TBST/BSA was added to each well. After incubating cells for 30 min at RT, wells were emptied and washed twice with TBST/BSA. 100 ul biotin-conjugated rabbit(Fab2) anti-mouse IgG (P/N315-066-046; Jackson Immunoresearch), diluted 1 :20,000 in TBST/BS, were added per well to stain the cells. After 30 min incubation at RT, wells were emptied and washed twice with

TBST/BSA. 100 ul Streptavidin-HRP conjugate (#21126; Pierce), diluted 1:20,000 in TBST/BSA, were added to each well and cells were incubated for 30 min at RT. Wells were washed twice with TBST/BSA. 100 ul of HRP substrate 3,3',5,5'-tetramethyl benzidine (#S1599; Dako Cytomation, Carpinteria, CA) were added. The reaction was stopped by adding 100 ul IN hydrochloric acid, usually after 20 min or when the desired staining intensity was reached. The enzymatic reaction was quantified by measuring the solution's absorbance at 450 nm wavelength.

Evaluation of supernatants from hybridomas in direct ELISA, sandwich ELISA and cell ELISA cells is shown in the table below. Values in the last column (column 7) are the ratio of OD values from cells transduced with Ovr232v3 (column 5) to cells transduced with alkaline phosphatase (column 6).

Table 5.

All D-series antibodies bound to recombinant Ovr232v3 (direct ELISA) and native Ovr232v3 on RK3E cells (cell ELISA). The comparison of antibodies binding to Ovr232v3 Constructs 7 and 2 shows that antibodies Dl, D5, D6, D28 and D31 bind to the v3-specific domain of Ovr232v3 (Gly26 - LeulOl).

Flow Cytometry Screening for Cell Surface Binding of Oyr232v3 MAbs - D Series

293F cells were transiently transfected with the expression plasmids Ovr232v3 Construct 4, EpCAM Construct 1 or a construct encoding Ovrl 10 (known in the literature as B7-H4) and hybridoma supernatants were.evaluated for cell surface staining as - — described above.

Table 6: Cell surface staining of Ovr232v3 transfected 293F cells with supernatants of uncloned h bridomas.

Antibodies D3, D4, D19, D20, D28, D30 and D31 preferentially bound to Ovr232v3-transfected cells as compared to EpCAM- or Ovrl 10-transfected cells indicating specific binding to Ovr232v3.

Cloning of Hybridomas Producing Oyr232v3 Specific MAb - D Series

The following hybridomas were cloned as described above: Dl, D3, D6, D7, D12, D13, D14, D16, D17, D23, D28, D30, D31 and D36. Antibodies were purified from supernatants of selected clones and re-analyzed by staining of Ovr232v3-transfected 293F cells and tumor cells. Results of binding to Ovr232v3-transfected 293F cells are in Table 7a and results of binding to tumor cells are in Table 7b.

Table 7a. Cell surface stainin of Oyr232v3 transfected 293F cells with urified MAbs .

All cloned D series hybridomas produce antibodies that preferentially bind to Ovr232v3-transfected 293F cells (Table 7a) and to Ovr232v3-positive tumor cell lines SW620 and HCTl 16 (Table 7b). Antibodies DLl, D3.1, D6.1 and D28.1 bind exclusively to Ovr232v3-transfected cells and do not bind to EpCAM- or Ovrl 10-transfected cells.

Oyr232v3 MAb Isotvpes

The isotypes of the anti-Ovr232v3 MAbs were determined using commercially available mouse monoclonal antibody isotyping immunoassay test kits (Iso Strip, Roche Diagnostic Corp., Indianapolis, IN). Results of the isotyping are listed in Table 8.

Oyr232v3 MAb Affinity Analysis

Binding kinetics and affinity constants were calculated from surface plasmon resonance measurements using a BIACORE 3000 instrument (Biacore, Piscataway, NJ). Experiments were designed to generate dissociation constants for the Ovr232v3 MAbs. Rabbit anti-mouse IgG antibody (Biacore) was immobilized on flow cells 2, 3, and

4 of a CM5 sensor chip (Biacore) by standard amine coupling (Biacore). Rabbit antipeptide antibody was coupled to flow cell one for reference subtractions. Ovr232v3 MAbs were captured on the rabbit anti-mouse-IgG coated chip, followed by binding of the antigen. Therefore these measurements represent real 1 : 1 affinities without avidity effect influences. Purified MAbs and hybridoma supernatants were diluted in HBS EP buffer. The Ovr232v3 MAbs were passed through the flow cells for 3 minutes at 20 uL/minute. The MAb capture level ranged between 100 and 1200 response units (RU) per flow cell. Following Ovr232v3 MAb capture the surface was allowed to stabilize for 2 minutes. The His-tagged Ovr232v3 protein encoded by Ovr232v3 Construct 6 was then flowed over the captured MAbs at 20 uL/minute in all flow cells, for 2 minutes, at a concentration of 10 ug/mL. A dissociation time of 300 seconds was allowed for data collection, and regenerations of the chip surfaces to anti-mouse IgG or anti-peptide IgG were performed by flowing 100 mM Glycine pH 2.0 through the flow cells for 30 seconds at 100 uL/minute. The resulting data were analyzed by BiaEvaluation software (Biacore) using a separate kd fit assuming Langmuir binding. During analysis, MAbs with antigen response of <60RU were dropped. The remainders were ranked by ascending kd:

Table 9: Ovr232v3 MAb Dissociation Constants

Western Blots

Protein extracts for western blot analysis were prepared in cell lysis buffer (1% NP-40, 1OmM Sodium Phosphate pH 7.2, 15OmM Sodium Chloride) from transfected RK3E-Ovr232v3, RK3E-Eρ-CAM and control RK3E-AP (alkaline phosphatase) cells. Proteins were separated by electrophoresis on NuPAGE 4-12% Bis-Tris gels (Invitrogen) under denaturing conditions in Novex-XCell II Minicell gel apparatus (Invitrogen) and subsequently transferred to PVDF membranes using an XCeIl II Blot Module (Invitrogen Life Technologies). Following the transfer of proteins, the membranes were blocked in PBST with 5% non fat milk at room temperature for at least an hour, followed by incubation overnight at 4°C with purified primary antibodies (Ovr232v3 monoclonal antibodies: C8.1, C9.1, C12.1, C14.1, C15.1, C16.1, C18.1, C20.1, C26.1, C29.1, C30.1, C31.1, C32.3, C59.1, C64.2, Dl.1, D3.1, D6.1, D7.1, D12.1, D13.1, D14.1, D16.1, D17.1, D23.1, D28.1, D31.1, and D36.1) and then with horseradish-peroxidase conjugated goat - anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, Inc.) and finally visualized by chemiluminesceiice using an ECL advance western blotting detection ldt (Amersham Biosiences, Piscataway, NJ). Tables 10a and 10b below summarize results of western blot experiments with anti-Ovr232v3 MAbs. Band intensity is categorized as weak (-), intermediate (+/-), strong (+), or not detected (ND). The size of the band(s) detected is also indicated.

Table 10a. Ovr232v3 MAb Western Blot Results- C series

As observed in Tables 10a and 10b, the Ovr232v3 MAbs C8.1, C9.1, C12.1, C14.1, C15.1, C16.1, C18.1, C26.1, C29.1, C30.1, C31.1, C59.1, D6.1, D7.1, D14.1, D16.T, D28.1, and D31.1 identified bands of the predicted size for Ovr232v3 protein (48 IcDa) in lysates of Ovr232v3 transduced RK3E cells.

Example 2: Epitope Mapping of Ovr232v3

The epitopes recognized by a panel of antibodies from the C and D series were determined by screening overlapping peptides for reactivity with the antibodies through an ELISA-based assay. Overlapping peptides were ordered from SynPep (Dublin, CA). The peptide sequences started at amino acid G21 in the N-terminus and ended at Al 11. These peptides span the v3 -specific region of the mature Ovr232v3 protein and the first 10 amino acids shared between Ovr232v3 and Ep-CAM. The peptides were provided in small aliquots with a range of 1-3 mg dissolved in 0.2 - 0.5 mL water. A 1 :400 dilution was made in PBS of each peptide and 50 μl were added to each well in duplicate on 96-well 4X Costar plates (#3690) (Costar Corporation; Cambridge, MA) and left overnight. The his-tagged extracellular domain of Ovr232v3 produced by transfection of 293T cells with Ovr232v3 Construct 6 was used as a positive control for each antibody. The next day, the plates were flicked dry and blocked with TBST 0.5% BSA for approximately 2 hours. Anti-Ovr232v3 antibodies (50 μl) were added either at 10 μg/ml per well (for purified antibodies) or as a 1:8 diluted hybridoma supernatant, and incubated at room temperature for approximately 2 hours. The plates were washed 3 times with TBST wash buffer. The secondary conjugate, goat anti-mouse Ig Fc-AP, (Pierce, Rockford, IL) was diluted 1 :5000 in a TBST/BSA solution and 50 μl was added to each well. The plates were shaken for 2 hours at room temperature. The plates were washed 3 times before 50 μl of substrate was added to each well and incubated for 35 minutes at room temperature. The substrate used was pNPP in IxDEA (1 mg/ml). To visualize the assay, plates were read at 405 nm on a SpectraMaxPlus (Molecular Devices, Sunnyvale, CA) using Softmax Pro (Molecular Devices, Sunnyvale, CA) and Excel (Microsoft; Seattle, WA) software for analysis. Table 11. e tides

Table 13. below summarizes the results of the anti-Oyr232v3 antibody epitope binding.

Anti-Ovr232v3 antibodies that bind peptides that overlap PTMs₅ motifs, or domains on Ovr232v3 are expected to block the function of the PTM, motif, domain or the biologic activity or function of Ovr232v3.

Example 3: Functional Validation of Ovr232v3

Surface expression of Oyr232v3 in transfected 293T cells

The coding sequences of human Ep-CAM and Ovr232v3 were obtained from colon cancer cDNA and subcloned into pCMV5His2 with c-terminal HA tags, described above as Ep-CAM Construct 1 and Ovr232v3 Construct 8. The plasmids were transiently transfected into 293 T cells using SuperFect transfection reagent (Qiagen, Valencia, CA). 48 hours later, the cells were washed and surface proteins were biotinylated with sulfo- NHS-SS-biotin (Pierce, Rockford, IL). Biotinylated proteins in the cell lysates were then captured by streptavidin agarose (Pierce, Rockford, IL). Both whole cell lysates and streptavidin pull down fractions were analyzed by SDS-PAGE and Western blot with antibody against the HA tag (Covance, Richmond, CA). Na+/K+ ATPase and GAPDH are shown as positive and negative controls for surface biotinylation, respectively. Both Ep- CAM and Ovr232v3 are detected as strong bands in the biotinylation fractions, indicating their presence on the cell surface. The results are summarized in the table below. Table 14.

Surface expression of Ovr232v3 was further confirmed by immunofluorescence in transduced RK3E cells. As described above, RK3E-Ovr232v3, RK3E-EpCAM, and RK3E-AP cells were generated then plated on coverslips 24 hours prior to surface staining. Cells were placed on ice and 5ug/ml of anti-Ep-CAM mAb C-10 (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the culture medium (DMEM + 10% FBS+ HEPES) for 30 min. After washing three times with IX PBS, cells were fixed with 4% formaldehyde, followed by addition of cy3 -conjugated donkey anti-mouse 2nd antibody (Jackson Immunoresearch Laboratories, West Grove, PA). The cells were then mounted on slides with Vectashield (with DAPI, Vector, Burlingame, CA) to visualize the cell nuclei and provide a counter stain, and observed in a Zeiss Fluorescence Microscope Axiophot equipped with the appropriate fluorescent filters. Micrographs were captured with a CCD camera. Both RK3E-Ovr232v3 and RK3E-EpCAM cells showed strong surface staining with the anti-Ep-CAM antibody. Control RK3E-AP cells did not bind to the primary antibody.

Both the biotynilation and immunofluorescence experiments demonstrate that like Ep-CAM, Ovr232v3 is localized to the cell surface and therefore is an ideal antibody therapeutic target.

Ep-CAM Specific Antibody Does Not Bind Oyr232v3 293T cells were transfected with either Ep-CAM or Ovr232v3 with C-terminal HA tags, as described above. Cell lysates were harvested in lysis buffer and run on SDS- PAGE under non-reducing conditions. Gels were transferred and the blots were probed with either anti-EpCAM mAb 323/A3 (Lab Vision, Fremont, CA) or anti-HA antibodies. The Anti-HA antibody recognized multiple bands from both Ep-CAM and Ovr232v3 transfectants, indicating the expression and multimerization of both proteins. On the other hand, anti-EpCAM 323/A3 mAb only recognized Ep-CAM and not Ovr232v3. This result suggests that even though Ovr232v3 contains all the peptide sequence present in Ep- CAM in addition to the Ovr232v3 unique region, differences in primary, secondary, and tertiary structures between these molecules result in exposure of different immunogenic epitopes. This demonstrates Ovr232v3 may be targeted independent of Ep-CAM with Ovr232v3 specific antagonists, specifically, ant-Ovr232v3 MAbs.

Growth Advantages of Oyr232v3 and Ep-CAM Expressing RK3E cells

Mϋnz et al (Oncogene, 2004 23:5748) have shown that expression of Ep-CAM in transfected HEK293 or NIH3T3 cells correlates with reduced growth factor requirement and increases in cell proliferation and colony formation. The growth rates of our RK3E- EpCAM, RK3E-Ovr232v3 and RK3E-AP (control) cells were measured in culture media (DMEM) containing either 10% or 1% FBS. Stably-transfected RK3E cells were seeded on 96 well plates at either 2000 or 1000 cells per well (day 0). Cell viability was determined by CelltiterGlo luminescence assay (Promega, Madison, WI) over the course of 4 days. Fold proliferation was calculated by normalizing luminescence units of all samples to the day 0 value of each cell type. At high starting cell density (2000 cells/well), no apparent growth difference was observed between RK3E-EpCAM, RK3E- Ovr232v3 and negative control RK3E-AP cells in either 10% or 1% FBS. However, at low starting cell density (1000cells/well), RK3E-EpCAM and RK3E-Ovr232v3 cells exhibited a growth advantage over control cells in 1% FBS media. The results are shown in the tables below.

Table 15. Transfected RK3E cell rowth with hi h startin cell densit 2000 cells/well)

As demonstrated above, expression of Ovr232v3 in tumor cell lines, like Ep-CAM, imparts a growth advantage to cells in low nutrient environments with sparse cell-cell contact. These conditions are similar to that of growing or metastatic tumors and expression of a cell surface antigen which imparts a growth advantage such as Ovr232v3 may contribute to tumor invasiveness, metastases establishment and survival. Therefore, inhibiting Ovr232v3 binding or function with an antagonist, such as an Ovr232v3 MAb, will suppress the growth advantage imparted to tumors by Ovr232v3 and limit or reverse tumor and metastases growth and survivability.

Qyr232v3 Expressing Cells are killed by anti-Ep-CAM MAb and 2nd Ab drug-conjugates

RK3E-Ovr232v3 Cells Killed by2nd antibody mAb-ZAP

RK3E-AP, RK3E-EpCAM and RK3E-Ovr232v3 cells were plated on 96 well plates and 0.4 ug/ml anti-EpCAM mAb C-10 (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the cells with or without 1 ug/ml of mAb-ZAP (Advanced Targeting Systems. San Diego, CA). Cell viability was measured by CelltiterGlo assay after 96 hours. The table below shows the percentages of viability after normalization to individual untreated control cells (medium alone). In the presence of the anti-EpCAM MAb, addition of mAb-ZAP caused 50% and 40% reduction of cell numbers in Ep-CAM and Ovr232v3 expressing cells, respectively. This cell killing is presumably mediated by internalization of these two proteins from the cell surface, as has been demonstrated with other targets and mAb-ZAP. Table 17.

SW620 Cells Killed by 2nd antibody Ab-saporin

SW620 cells, which natively express Ep-CAM and Ovr232v3 mRNA (detected by Quantitative RT-PCR), were utilized to evaluate cell killing effects of anti-EpCAM antibodies alone or in combination with a drug-conjugated 2^nd Ab. SW620 cells were treated with 0.4 ug/ml anti-CD71 antibody 5E9 (ATCC₅ Manassus, VA), anti-EpCAM antibody C-IO (Santa Cruz Biotechnology) or negative control mAb anti-ricin antibody TFTBl (ATCC, Manassus, VA) with or without 1 ug/ml of mAb-ZAP as described above. CD71 and ricin were used as positive and negative controls, respectively. Anti-EpCAM induced 30% killing in native tumor cells SW620 while anti-TfiiR eliminated > 96% of cells due to the rapid internalization and recycling of the receptors. Similar results were obtained in another tumor cell line, MCF-7. Table 18.

These data show that cells overexpressing Ovr232v3 or Ep-CAM are efficiently killed by binding of a primary MAb which is bound by a 2^nd conjugate Ab, presumably via internalization of the target. The results further show that results are not unique to engineered cells, but consistent with native tumor cells. This demonstrates that Ovr232v3 expressing tumor cells can be killed via a target specific MAb and 2^nd drug conjugate antibody in vivo.

Example 4: siRNA Knockdown of Ovr232v3 siRNA Oligonucleotide Design and Preparation

To design Ovr232v3 specific siRNA molecules, sequences were selected from the unique region of the Ovr232v3 mRNA to ensure the sequence will not affect Ep-CAM expression based on the vendor's designing tool (block-iT™ RNAi Designer, Invitrogen Inc. Carlsbad, CA). Methods for designing siRNA molecules have been described previously as well (Elbashir et al, 2001, Nature 411 :494-498). Stealth™ siRNAs for Ovr232v3, Ep-CAM and RNAi Negative Control Med GC (cat # 12935-300) were purchased from Invitrogen. The negative control siRNA should not generate knockdown of any known cellular mRNA. A BLAST search against the human genome was performed with each selected siRNA sequence to ensure that the siRNA was target- specific and would not function to knockdown other sequences. Ep-CAM siRNA was designed using the same procedure, however, knockdown of Ovr232v3 with Ep-CAM siRNA was expected due to the homology between Ep-CAM mRNA and Ovr232v3 mRNA. Ovr232v3_siRNA-l sense 5'-GGGAGCAGCCUCACUUCGCAGCUUU-S' (SEQ ID NO: 33) Ovr232v3_siRNA-2 sense 5'-CCUCACUUCGCAGCUUUGCUCGCCU-S' (SEQ ID NO: 34) Ovr232v3_siRNA-3 sense 5'-GGAUUGGAGCAGAGUUGUG-S' (SEQ ID NO: 35) Ovr232v3_siRNA-4 sense 5'-ACGGGCAUAAUAGGGAGGG-S' (SEQ ID NO: 36) Ovr232v3_siRNA-5 sense 5'-UAGGGAGGGGACCAAGAGG-S' (SEQ ID NO: 37) Ovr232v3_siRNA-6 sense 5'-GAGGCCGCGCUUUCCAGCG-S' (SEQ ID NO: 38) Ovr232v3_siRNA-7 sense 5'-ACGGCGAGGGCCGUCCCGG-S' (SEQ ID NO: 39) Ep-CAM_siRNA sense 5'-GCUCAGGAAGAAUGUGUCUGUGAAA-S' (SEQ ID NO: 40)

Ovr232v3_siRNA-l, Ovr232v3_siRNA-2 and Ep-CAM_siRNA siRNA molecules were chemically synthesized by Invitrogen Inc. The siRNA oligonucleotides were resuspended with DEPC-treated water to yield a 20microM solution.

Transfection with siRNA Oligonucleotides

The above siRNAs were first tested on HCTl 16-Ovr232v3 cells to check their efficiency in silencing Ovr232v3 and Ep-CAM. RNA oligonucleotides were mixed with 7.8uL transfection reagent Hi-Perfect (Qiagen, Valencia, CA) for 10 min before being added to 24,000 cells. The whole mixture was then plated on 6 well plates for 5 hours until cells are attached. Fresh media were then added and cells were incubated at a 37 degree incubator for 3 days. Lysates were harvested for Western blot analysis of Ep-CAM and Ovr232v3 signals with Ep-CAM mAb ClO (Santa Cruz Biotechnology, Santa Cruz, CA) and Ovr232v3 mAb C15.1, respectively. Table 19 summarizes the result of protein expression levels detected after knockdown. Table 19.

The above results demonstrate that by targeting the Ovr232v3 -specific sequence Ovr232v3 specific siRNA such as Ovr232v3 siRNA- 1 and Ovr232v3 siRNA-2 can efficiently and specifically knockdown Ovr232v3 expression.

For proliferation assays, HCTl 16 which express Ovr232v3 and MG63 cells which do not express Ovr232v3 were evaluated. First, 2000 HCTl 16 cells and 1500 MG63 cells were each mixed with 1OnM siRNA and 0.13 uL transfection reagent Hi-Perfect and then seeded on 96-well plates. Cell proliferation was measured by CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) over a 5 day period. Fold proliferation was calculated by normalizing each luminescence value to the reading of day 0.

Table 20. siRNA treatment in cell line HCTl 16

Results In Ovr232v3 positive HCTl 16 cells, treatment of Ovr232v3 siRNAs for 5 days- - greatly reduced cell proliferation by 82-88% compared to no siRNA samples. Ep-CAM siRNA knockdown also inhibited cell growth by 91%. Conversely, the Ovr232v3 siRNAs had no or minimal effects on Ovr232v3 negative cell line MG-63. These assays confirmed that Ovr232v3 is essential to cell proliferation and that reduction of expression or inhibition of function with anti-Ovr232v3 antibodies is useful for inhibition of cellular growth.

Example 5: Anti-Ovr232v3 MAbs are internalized into Ovr232v3 positive cells

The following cancer cell lines were used in this study and were obtained from the ATCC: colon (HCTl 16), colon (SW620) and cervical (HeLa). HCTl 16 and SW620 cell lines express Ovr232v3 RNA as determined by QPCR. HeLa does not express Ovr232v3 RNA.

The above cell lines were seeded onto sterile 12 mm glass coverslips and cultured at 37⁰C in DMEM/10% FBS for 48 hr prior to treatment with the primary antibodies (Ovr232v3 MAbs). MAbs Ovr232v3.C9.1, Ovr232v3.C12.1, and Ovr232v3.C64.2 were tested by immunofluorescence microscopy to determine which of these antibodies bound specifically to the Ovr232v3 expressing cancer cells and were internalized. Primary MAbs were added to the medium at a final concentration of 5ug/ml and incubated for one hour at 37⁰C. Following fixation with 4% formaldehyde in Phosphate Buffered Saline (PBS), the cells were incubated with a secondary Cy3 -labeled donkey anti-mouse antibody (Jackson Inimunoresearch Laboratories, West Grove, PA) at a concentration of 5ug/ml for 30 min. Following washing, the cells were mounted in Vectashield (Vector, Burlingame, CA), a medium containing DAPI to visualize the cell nuclei and observed in a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with the appropriate fluorescent filters. Micrographs were recorded using a CCD camera.

Ovr232v3.C9.1, Ovr232v3.C12.1, and Ovr232v3.C64.2 all bound to Ovr232v3 expressing cells. Figures 1, 2 and 3 demonstrate the binding and internalization of the above MAbs into HCTl 16 (Fig. 1) and SW620 cells (Fig. 2) but not Ovr232v3 negative cell HeLa (Fig. 3). Most of the cells in the field showed staining of Ovr232v3.C9.1 (Fig IA₃ Fig. 2A), Ovr232v3.C12.1 (Fig IB, Fig. 2B), and Ovr232v3.C64.2 (Fig 1C, Fig. 2C) in the vesicular structures inside the cells, while no staining was observed in negative control HeLa cells (Fig 3A, Fig 3B, Fig 3C). Figures ID, 2D and 3D are negative controls demonstrating that the secondary labeled antibody does not bind the cells and is not internalized without Ovr232v3 antibodies. These results indicate anti-Ovr232v3 antibodies, and in particular are suitable for immunotherapy of tumors with or without conjugated drugs, toxins, enzymes, prodrug activating molecules or isotopes.

Example 6: Anti-Ovr232v3 MAbs and conjugated 2^nd MAb kill Ovr232v3 expressing cells RK3E-Ovr232v3, RK3E-Eρ-CAM and control RK3E-AP cells were generated as described above and incubated with anti-Ovr232v3 MAbs premixed with MAb-ZAP goat anti-mouse Ig saporin conjugate (Advanced Targeting Systems, San Diego, CA). Cell viability was measured to detect killing effects of anti-Ovr232v3 MAbs with 2^nd conjugate antibody on Ovr232v3 expressing cells. The anti-rat -transferin receptor MAb (BD Biosciences Pharmingen, San Jose, CA) and the anti-ricin MAb TFTBl (ATCC, Manassus, VA) were used as positive and negative control MAbs for cell killing, respectively.

RK3E-Ovr232v3, RK3E-Ep-CAM and RK3E-AP cells were placed into 96 well, flat bottom, sterile white cell culture plates (Coming), in triplicate wells, at 2000 cells/100 uL /well, in DMEM medium with 10%FBS. Plates were incubated at 37⁰C, in 5% CO2 for 2 hours to allow cells to attach (day 0). 25 uL of 5x final MAb concentrations alone, or 25 uL of 5x MAb premixed with 25 uL of 5x MAb-ZAP, or 25 uL of 5x Mab Zap alone, or 25 uL of medium alone were added to wells of the 96 well plates, in triplicate, to a final volume of 125 uL. Final MAb concentrations were 2 ug/mL, 0.4 ug/mL, 0.08 ug/mL and 0 ug/mL and the final concentration of MAb-ZAP was 1 ug/mL. Triplicate wells with medium alone, MAb alone, and MAb-ZAP alone were used as negative controls. On day 4, cell viability was measured by CellTiterGlo Luminescent Cell Viability assay (Promega. Madison. WI). Percentage of cell growth was calculated by normalizing the luminescence unit of samples to medium alone (100%) on each plate. For tumor cell killing experiment, HCTl 16 and HeLa cells were plated on 96 well plates at 2000 and 1500 cells per well, respectively. Final MAb concentrations were 2 ug/mL, 1.0 ug/mL, 0.5 ug/mL and 0 ug/mL and the final concentration of MAb-ZAP was 1 ug/mL. Anti- human transferring receptor 5E9 (anti-CD71, ATCC, Manassus, VA) and anti-ricin were used as positive and negative control MAbs for cell killing.

Table 22. RK3E-Ovr232v3 killing by C-series Ovr232v3 MAbs and MAb-ZAP Saporin Conjugate

Table 23. RK3E-AP killing by C-Series Ovr232v3 MAbs and MAb-ZAP Saporin Conjugate ^

Table 24, -RK3E-Ovr232v3 killing by D-series Ovr232v3 MAbs and MAb-ZAP Saporin Conjugate

Table 25. RK3E-EpCAM killing by D-series Ovr232v3 MAbs and MAb-ZAP Saporin Conjugate

Table 26. RK3E-AP killing by D-series Ovr232v3 MAbs and MAb-ZAP Saporin Conu ate

Table 27. HCTl 16 killin b Oyr232v3 MAbs and MAb-ZAP Sa orin Conu ate

Table 28. HeLa killing by Ovr232v3 MAbs and MAb-ZAP Saporin Conjugate

Results of testing Ovr232v3.C8.1, Ovr232v3.C9.1, Ovr232v3.C12.1, Ovr232v3.C14.1, Ovr232v3.C15.1, Ovr232v3.C16.1, Ovr232v3.C18.1, Ovr232v3.C20.1₅ Ovr232v3.C26.1, Ovr232v3.C29.1, Ovr232v3.C30.1, Ovr232v3.C31.1, Ovr232v3.C32.3, Ovr232v3.C59.1, Ovr232v3.C64.2, Ovr232v3.Dl.l, Ovr232v3.D3.1, Ovr232v3.D6.1, Ovr232v3.D13.1, Ovr232v3.D17.1, Ovr232v3.D28.1, Ovr232v3.D30.1, Ovr232v3.D31.1 and Ovr232v3.D36.1 are presented in Tables 22-28 above. The mAb-ZAP alone did not inhibit growth of Ovr232v3, Ep-CAM or AP expressing cells, demonstrating that non- internalized saporin conjugate alone was not responsible for cell death. However, In the presence of lug/ml of mAb-ZAP, Ovr232v3 mAbs (at 0.4 ug/ml) Ovr232v3.C8.1,

Ovr232v3.C9.1, Ovr232v3.C12.1, Ovr232v3.C14.1₅ Ovr232v3.C15.1, Ovr232v3.C16.1, Ovr232v3.C18.1, Ovr232v3.C20.1, Ovr232v3.C29.1, Ovr232v3.C31.1, Ovr232v3.C32.3, Ovr232v3.C59.1, and Ovr232v3.C64.2 caused 40-95% growth inhibition of Ovr232v3 expressing cells, but not in control AP expressing cells. In comparison, positive control anti-TfhR MAb inhibited 99.9% of cell growth (at 0.4 ug/ml). Among the D series mAbs, at 0.4ug/ml,_Ovr232v3.Dl.l, Ovr232v3.D3.1, Ovr232v3.Dβ.l, Ovr232v3.D13.1, ^~

Ovr232v3.D17.1, Ovr232v3.D28.1, Ovr232v3.D30.1, Ovr232v3.D31.1 and Ovr232v3.D36.1 inhibited RK3E-Ovr232v3 cell growth from 25% to 93% but had no effect on either RIGE-Ep-CAM or RK3E-AP cells. Notably, the anti-Ep-CAM MAb (clone 323/A3, Lab Vision, CA) only inhibited cell growth of RK3E-Ep-CAM but not RK3E-Ovr232v3 or RK3E-AP cells.

Anti-Ovr232v3 antibodies Ovr232v3.C8.1, Ovr232v3.C9.1, Ovr232v3.C12.1, Ovr232v3.C14.1, Ovr232v3.C15.1, Ovr232v3.C16.1, Ovr232v3.C18.1, Ovr232v3.C20.1, Ovr232v3.C26.1, Ovr232v3.C29.1, Ovr232v3.C30.1, Ovr232v3.C31.1, Ovr232v3.C32.3, Ovr232v3.C59.1 and Ovr232v3.C64.2 were generated with peptide specific to Ovr232v3 as described above, therefore specifically bind Ovr232v3 and not Ep-CAM.

Additionally, antibodies were tested on RK3 E-Ep-CAM to examine their potential cross-reactivity to Ep-CAM. The following antibodies did not show killing activity to RK3E-Ep-CAM cells: Ovr232v3.Dl.l, Ovr232v3.D3.1, Ovr232v3.D6.1, Ovr232v3.D13.1, Ovr232v3.D17.1, Ovr232v3.D28.1, Ovr232v3.D30.1, Ovr232v3.D31.1 and Ovr232v3.D36.1. This result indicates that the above D series mAbs also preferentially interact with Ovr232v3. The ability of these antibodies to specifically bind Ovr232v3 demonstrates usefulness in a therapeutic setting to specifically target and kill Ovr232v3 -expressing tumors in vivo. When tumor cells HCTl 16 which natively express Ovr232v3 were used in the niAb-ZAP assay, MAbs C64.2, D13.1 and D36.1 produced 10- 26% growth inhibition and had no effect on the negative control HeLa cells (Tables 27 and 28).

The ability of these antibodies to preferentially bind Ovr232v3 demonstrates usefulness in a therapeutic setting to specifically target and kill Ovr232 v3 -expressing tumors in vivo with or without conjugated drugs, toxins, enzymes, prodrug activating molecules or isotopes.

Example 7: Deposits

Deposit of Cell Lines and DNA

The following hybridoma cell lines were deposited with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110-2209, U.S.A., and accorded accession numbers.

Table 29: ATCC deposits

Anti-Ovr232v3 antibody hybridomas Ovr232v3.C31.1 and Ovr232v3.C32.3 were shipped to the ATCC via FedEx Overnight on 28 July 2005. The Tracking number for the shipment was 850827731952. FedEx confirmed delivery to ATCC on 29 July 2005 by email. Additionally, a Patent Specialist at ATCC confirmed receipt of the hybridoma deposits in good condition on 29 July 2005. Anti-Ovr232v3 antibody hybridomas Ovr232v3.C31.1 and Ovr232v3.C32.3 were given deposit accessions PTA-6894 and PTA- 6895, respectively. The names of the deposited hybridoma cell lines above may be shortened for convenience of reference. E.g. A57.1 corresponds to Ovr232v3.A57.1. These hybridomas correspond to the clones (with their full names) listed in Table 29.

These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations there under (Budapest Treaty). This assures maintenance of viable cultures for 30 years from the date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between diaDexus, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the cultures to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 3 7 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultures on deposit should die or be lost or destroyed when cultivated under suitable conditions, they will be promptly replaced on notification with a viable specimen of the same culture. Availability of the deposited strains are not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The making of these deposits is by no means an admission that deposits are required to enable the invention. ,. . . . . .

Claims

We Claim:

I . An isolated Ovr232v3 antibody that selectively binds a mammalian cell.

2. The antibody of claim 1 which internalizes upon binding.

3. The antibody of claim 1 wherein the mammalian cell is a living cell.

4. The antibody of claim 1 which is a monoclonal antibody.

5. The antibody of claim 1 which is an antibody fragment.

6. The antibody of claim 1 which is a chimeric or a humanized antibody.

7. The antibody of claim 1 which binds an Ovr232v3 peptide consisting of Gly26 to Leul02 ofOvr232v3.

8. The antibody of claim 7 wherein the Ovr232v3 peptide contains a post translational modification, motif, or domain.

9. The antibody of claim 8 wherein the post translational modification, motif, or domain is a phosphorylation, myristoylation, RGD integrin binding motif, or a Pro-Arg rich protein binding motif.

10. The antibody of claim 1 where the antibody binds to the epitope comprising SEQ ID NO: 22-29.

I 1. The antibody of claim 4 which is produced by a hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

12. The antibody of claim 4, wherein the antibody competes for binding to the same epitope as the epitope bound by the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

13. The antibody of claim 4 which is conjugated to a growth inhibitory agent.

14. The antibody of claim 4 which is conjugated to a cytotoxic agent.

15. The antibody of claim 14 wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

16. The antibody of claim 15 wherein the cytotoxic agent is a toxin.

17. The antibody of claim 16, wherein the toxin is selected from the group consisting of ricin, saponin, maytansinoid and calicheamicin.

18. The antibody of claim 11, wherein the toxin is a maytansinoid.

19. The antibody of claim 4, wherein the mammalian cell is a cancer cell.

20. An isolated anti-Ovr232v3 monoclonal antibody that selectively binds an Ovr232 v3 -expressing cell.

21. An isolated anti-Ovr232v3 monoclonal antibody that inhibits the growth of Ovr232v3-expressing cancer cells.

22. The antibody of claim 21 which is a humanized or human antibody.

23. The antibody of claim 22 which is produced by bacterial, insect, or mammalian cells.

24. The antibody of claim 20, which is a humanized form of an anti-Ovr232v3 antibody produced by a hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

25. The antibody of claim 21, wherein the cancer cells are from a cancer selected from the group consisting of ovarian, colon, prostate, and lung cancer.

26. The antibody of claim 21, wherein the cancer is ovarian, colon, prostate, or lung cancer.

27. A cell that produces the antibody of claim 4.

28. The cell of claim 27, wherein the cell is selected from the group consisting of a hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

29. A method of producing the antibody of claim 4 comprising culturing an appropriate cell and recovering the antibody from the cell culture.

30. A composition comprising the antibody of claim 4 or claim 20, and a carrier.

31. The composition of claim 30, wherein the antibody is conjugated to a cytotoxic agent.

32. The composition of claim 31 , wherein the cytotoxic agent is a maytansinoid.

33. The composition of claim 30, wherein the antibody is a human or humanized antibody and the carrier is a pharmaceutical carrier.

34. The composition of claim 33, wherein the humanized antibody is a humanized form of an anti-Ovr232v3 antibody produced by hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

35. A method of killing an Ovr232v3-expressing cancer cell, comprising contacting the cancer cell with the antibody of claim 1 or claim 2, thereby killing the cancer cell.

36. The method of claim 35, wherein the cancer cell is selected from the group consisting of an ovarian, colon, prostate, and lung cancer cell.

37. The method of claim 36, wherein the cancer cell is an ovarian or colon cancer cell.

38. The method of claim 36, wherein the cancer cell is from metastatic ovarian, colon, prostate, or lung cancer.

39. The method of claim 35, wherein the antibody is an antibody fragment.

40. The method of claim 35, wherein the antibody is a humanized antibody.

41. The method of claim 35, wherein the antibody is conjugated to a cytotoxic agent.

42. The method of claim 41 , wherein the cytotoxic agent is a toxin selected from the group consisting of maytansinoid, ricin, saporin and calicheamicin.

43. The method of claim 41 , wherein the cytotoxic agent is a radioactive isotope

44. The method of claim 35, wherein the antibody is a humanized form of the antibody produced by hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

45. A method of alleviating an Ovr232v3-expressing cancer in a mammal, comprising administering a therapeutically effective amount of the antibody of claim 20 to the mammal.

46. The method of claim 45, wherein the cancer is selected from the group consisting of ovarian, colon, prostate, and lung cancer.

47. The method of claim 45, wherein the antibody is a humanized antibody.

48. The method of claim 45, wherein the antibody is conjugated to a cytotoxic agent.

49. The method of claim 48, wherein the cytotoxic agent is a maytansinoid.

50. The method of claim 49, wherein the antibody is administered in conjunction with at least one chemotherapeutic agent.

51. The method of claim 50 wherein the chemotherapeutic agent is paclitaxel or derivatives thereof.

52. An article of manufacture comprising a container and a composition contained therein, wherein the composition comprises an antibody of claim 4.

53. The article of manufacture of claim 52 further comprising a package insert indicating that the composition can be used to treat ovarian, colon, prostate, or lung cancer.

54. A method for determining if cells in a sample express Ovr232v3 comprising (a.) contacting a sample of cells with an Ovr232v3 antibody of claim 4 under conditions suitable for specific binding of the Ovr232v3 antibody to Ovr232v3, and

(b.) determining the level of binding of the antibody to cells in the sample, or the level of Ovr232v3 antibody internalization by cells in said sample, W

154

wherein Ovr232v3 antibody binding to cells in the sample or internalization of the Ovr232v3 antibody by cells in the sample indicate cells in the sample express Ovr232v3.

55. The method of claim 54 wherein said sample of cells are contacted with an antibody produced by a hybridoma deposited with the American Type Culture Collection on 29 July 2005 selected from the group consisting of Ovr232v3.C31.1 and Ovr232v3.C32.3.

56. The method of claim 54 wherein said sample of cells is from a subject who has a cancer, is suspected of having a cancer or who may have a predisposition for developing cancer.

57. The method of claim 56 wherein the cancer is a ovarian, colon, prostate, or lung cancer.

58. The method of claim 54 wherein said antibody is a labeled antibody. . - -

59. A method for detecting Ovr232v3 overexpression in a test cell sample, comprising: (a.) combining a test cell sample with an Ovr232v3 antibody of claim 4 under conditions suitable for specific binding of the Ovr232v3 antibody to Ovr232v3 expressed by cells in said test sample, (b) determining the level of binding of the Ovr232v3 antibody to the cells in the test sample, and (c) comparing the level of Ovr232v3 antibody bound to the cells in step (b) to the level of Ovr232v3 antibody binding to cells in a control cell sample, wherein an increase in the binding of the Ovr232v3 antibody in the test cell sample as compared to the control is indicative of Ovr232v3 overexpression by cells in the test cell sample.

60. The method of claim 59 wherein the test cell sample is a cancer cell sample.

61. The method of claim 60 wherein the cancer cell sample is of a ovarian, colon, prostate, or lung cancer.

62. The method of claim 61 wherein the ovarian, colon, prostate, or lung cancer is metastatic ovarian, colon, prostate, or lung cancer.

63. The method of claim 60 wherein the control is a sample of adjacent normal tissue.

64. A method for detecting Ovr232v3 overexpression in a subject in need thereof comprising,

(a.) combining a bodily fluid sample of a subject with an Ovr232v3 antibody of claim 4 under conditions suitable for specific binding of the Ovr232v3 antibody to Ovr232v3 in said bodily fluid sample,

(b.) determining the level of Ovr232v3 in the bodily fluid sample, and (c.) comparing the level of Ovr232v3 determined in step b to the level of

Ovr232v3 in a control bodily fluid sample, wherein an increase in the level of Ovr232v3 in the bodily fluid sample from the subject as compared to the control is indicative of Ovr232v3 overexpression in the subject.

65. The method of claim 64 wherein the subject has cancer.

66. The method of claim 65 wherein the subject has ovarian, colon, prostate, or lung cancer.

67. The method of claim 66 wherein the ovarian, colon, prostate, or lung cancer is metastatic ovarian, colon, prostate, or lung cancer.

68. The method of claim 64, wherein the bodily fluid is selected from the group consisting of blood, serum, plasma, urine, ascites, peritoneal wash, saliva, sputum, seminal fluids, mucous membrane secretions, and other bodily excretions such as stool.

69. The method of claim 64 wherein the control is a bodily fluid sample from a subject without a cancer overexpressing Ovr232v3.

70. , A screening method for antibodies that bind to an epitope which is bound by an antibody of claim 4 comprising, (a.) combining an Ovr232v3 -containing sample with a test antibody and an antibody of claim 4 to form a mixture ,

(b.) determining the level of Ovr232v3 antibody bound to Ovr232v3 in the mixture, and

(c.) comparing the level of Ovr232v3 antibody bound in the mixture of step (a) to a control mixture, wherein the level of Ovr232v3 antibody binding to Ovr232v3 in the mixture as compared to the control is indicative of the test antibody's binding to an epitope that is bound by the anti-Ovr232v3 antibody of claim 4.

71. The screening method of claim 70_. wherein the level of Ovr232v3 antibody bound to Ovr232v3 is determined by ELISA or flow cytometry.

72. The screening method of claim 70 wherein the control is a mixture of Ovr232v3, Ovr232v3 antibody of claim 4 and an antibody known to bind the epitope bound by the Ovr232v3 antibody of claim 4.

73. The screening method of claim 70 wherein the anti-Ovr232v3 antibody is labeled.

74. The screening method of claim 73 wherein the Ovr232v3 is bound to a solid support.

75. The screening method of claim 74 wherein the solid support is a sepharose bead.