Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
  • A61K47/6819—Plant toxins
  • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
• • A—HUMAN NECESSITIES
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  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1054—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from lung
• • A—HUMAN NECESSITIES
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  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1057—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from liver or pancreas
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/303—Liver or Pancreas
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3046—Stomach, Intestines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell

Definitions

• the present invention relates to anti-Ovrl 15 antibody compositions and methods of killing Ovrl 15-expressing ovarian, pancreatic and colon cancers cells.
• 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.
• the American Cancer Society estimates that there will be about 25,580 new cases of ovarian cancer in 2004 in the United States alone. Ovarian cancer will cause about 16,090 deaths in the United States in the same year.
• ACS Website cancer with the extention .org of the world wide web.
• BRCA1 located on chromosome 17, and BRCA2, 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.
• 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.
• 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.
• 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 mmor 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, mmor 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.
• Substage IIA involves extension and/or implants on the uterus and/or fallopian tubes, with no malignant cells in the ascites or peritoneal washings, while substage IIB involves extension into other pelvic organs and tissues, again with no malignant cells in the ascites or peritoneal washings.
• Substage IIC involves pelvic extension as in IIA or IIB, but with malignant cells in the ascites or peritoneal washings.
• Stage III ovarian cancer involves tumor growth in one or both ovaries, with peritoneal metastasis beyond the pelvis confirmed by microscope and/or metastasis in the regional lymph nodes.
• Substage IIIA is characterized by microscopic peritoneal metastasis outside the pelvis, with substage IIIB involving macroscopic peritoneal metastasis outside the pelvis 2 cm or less in greatest dimension.
• Substage IIIC is identical to IIIB, except that the metastasis is greater than 2 cm in greatest dimension and may include regional lymph node metastasis.
• Stage IV refers to the presence distant metastasis, excluding peritoneal metastasis. Id.
• 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, m. Ovarian Cancer 241 (Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001).
• treatment typically consists of: (1) cytoreductive surgery, including total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and lymphadenectomy, followed by (2) adjuvant chemotherapy with paclitaxel and either cisplatin or carboplatin.
• Pancreatic cancer is the thirteenth-most common cancer and eighth-most cause of cancer death worldwide. Donghui Li, Molecular Epidemiology, in Pancreatic Cancer 3 (Douglas B. Evans et al. eds., 2002). In the United States, cancer of the pancreas is the fourth-most common cancer in both males and females, accounting for five percent of cancer deaths and nearly 30,000 deaths overall. Id. The rates of pancreatic cancer are higher in men than women and higher in African-Americans as opposed to Caucasians. Id. at 9. The most significant predictor of pancreatic cancer is patient age; among Caucasians, the age-related incidence of pancreatic cancer increases continuously, even through the 85 and older category. Id. at 3.
• pancreatic cancer Aside from age, a number of risk factors for pancreatic cancer have been identified, including smoking, diet, occupation, certain medical conditions, heredity, and molecular biologic. Smoking is the most important risk factor for acquiring the disease, with the link between smoking and pancreatic cancer being established in numerous studies. Li, supra at 3. The relative risk amounts to at least 1.5, increasing with the level of smoking to an outer risk ratio of 10-fold. Id. The next most important factor would appear to be diet, with increased risk associated with animal protein and fat intake, and decreased risk associated with intake of fruits and vegetables. Id. at 3-4. As for particular occupations, excessive rates of pancreatic cancer have been associated with workers in chemistry, coal and gas exploration, the metal industry, leather tanning, textiles, aluminum milling, and transportation. Id.
• pancreatic cancer A number of medical conditions have also been associated with an increased incidence of pancreatic cancer, including diabetes, chronic pancreatitis, gastrectomy, and cholecystectomy, although the cause and effect relationship between these conditions and pancreatic cancer has not been established. Id.
• Hereditary genetic factors comprise less than 10% of the pancreatic cancer burden, with associations documented with hereditary pancreatitis, as well as germline mutations in familial cancer syndrome genes such as hMSH2 and hMLHl (hereditary nonpolyposis colon cancer), pi 6 (familial atypical multiple mole-melanoma) and BRCA1/BRCA2 (breast and ovarian cancer). Id. at 3. While no other organ has a higher inherited basis for cancer than the pancreas, researchers have been unable to pinpoint the particular genetic defect(s) that contribute to one's susceptibility to pancreatic cancer. David H. Berger & William E. Fisher, Inherited Pancreatic Cancer Syndromes, in Pancreatic Cancer 73 (Douglas B. Evans et al. eds., 2002).
• pancreatic cancer From the standpoint of molecular biology, research has revealed an association between pancreatic cancer and a number of genetic mutations, including the activation of the proto-oncogene K-ras and the inactivation of the tumor suppressor genes p53,p!6, and DPC4. Marina E. Jean et al, The Molecular Biology of Pancreatic Cancer, in Pancreatic Cancer 15 (Douglas B. Evans et al. eds., 2002).
• pancreatic adenocarcinomas 83% possessed K-ras activation along with inactivation of pl6 and p53.
• K-ras mutations are found in 80 to 95% of pancreatic adenocarcinomas, with p53, pi 6, and DPC4 genes being the must frequently deleted tumor suppressor genes in cancer of the pancreas. Howe, supra at 29. Homozygous deletions, hypermethylation, and mutations of the pi 6 gene have been discovered in 85 to 98% of adenocarcinomas of the pancreas. Id.
• pancreatic cancer is particularly essential for this deadly cancer, as most patients fail to present until their pancreatic tumors obstruct the bile duct or induce pain, at which point the tumors have invaded the capillary and lymphatic vessels that surround the pancreas, Howe, supra at 29; unfortunately, patients with the metastatic form of the disease typically survive less than one year after diagnosis, Jean et ah, supra at 15. While computed tomography (CT) and endoscopic retrograde cholangiopancreatography (ERCP) may assist in the diagnosis of symptomatic patients, there is presently no tool for screening for pancreatic tumors that would permit their early discovery, at which point they might be curable. Howe, supra at 29.
• CT computed tomography
• ERCP endoscopic retrograde cholangiopancreatography
• Markers such as carcinoembryonic antigen, and antibodies generated against cell lines of human colonic cancer (CA 19-9 and CA 195), human ovarian cancer (CA 125), and human pancreatic cancer (SPAN-1 and DUPAN-2) may be elevated in the sem of patients with pancreatic cancer, but these markers are not sufficiently reliable to serve as screening tools due to their lack of specificity and appearance late in the disease.
• pancreatic cancer Once pancreatic cancer has been diagnosed, treatment decisions are made in reference to the stage of cancer progression.
• a number of imaging techniques are employed to stage pancreatic cancer, with computed tomography (CT) being the present method of choice, Harmeet Kaur et al, Pancreatic Cancer: Radiologic Staging, in Pancreatic Cancer 86 (Douglas B. Evans et al. eds., 2002); Ishiguchi, T. et al, Hepatogastroenterology 48(40): 923-27 (2001), despite the fact that it frequently underestimates the extent of the cancer, as small- volume metastases are often beyond the resolution of CT, H. J. Kim & K. C.
• MRI may at some point supplant CT in view of, inter alia, its ability to (1) contrast among various tissue, (2) modify pulse sequences to improve visualization of lesions and minimize artifacts, (3) perform imaging while limiting a patient's exposure to ionizing radiation, and (4) visualize vessels without using IV iodinated contrast reagents.
• MRI has not demonstrated a clear advantage over CT.
• TUS transabdominal ultrasound
• EUS endoscopic ultrasound
• IUS intraoperative ultrasound
• TUS is hindered by gas in the gastrointestinal tract and fat in the peritoneum
• EUS requires considerable experience in ultrasonography and endoscopy and may not be widely available
• IUS can only be used intraoperatively.
• TNM TNM
• N regional lymph nodes
• M distant metastasis
• Stage 0 is characterized by carcinoma in situ (Tis), with no regional lymph node metastasis (NO) and no distant metastasis (M0). Id. at 113. Stages I and II differ from stage 0 only in terms of tumor category: stage I involves a tumor limited only to the pancreas that is either (1) 2 cm or less in greatest dimension (TI) or (2) more than 2 cm in greatest dimension (T2), while stage II involves a tumor that extends directly into the duodenum, bile duct, or peripancreatic tissues (T3). Id.
• Stage III involves tumor category TI, T2, or T3; regional lymph node metastasis (Nl), which involves either a single lymph node (pNla) or multiple lymph nodes (pNlb); and no distant metastasis (M0).
• Stage IVA is characterized by tumor extension directly into the stomach, spleen, colon, or adjacent large vessels (T4); any N category; and no distant metastasis (M0).
• stage IVB is characterized by any T category, any N category, and distant metastasis (Ml). Id.
• chemotherapeutic agents such as gemcitabine and 5- fluorouracil have shown some effectiveness against pancreatic carcinomas, the reality is that chemotherapy has shown little impact on survival from pancreatic cancer. Burdette, supra at 101. Radiation therapy has provided conflicting results with respect to its efficacy, id., although radiation in combination with 5-fluorouracil has shown some promise, Regine, supra at 235.
• pancreatic cancer for diagnosing pancreatic cancer, for monitoring the progression of the disease, for staging pancreatic cancer, for determining whether pancreatic cancer has metastasized, for imaging pancreatic cancer and for better treatment of pancreatic 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).
• the American Cancer Society estimates that there will be about 106,370 new cases of colon cancer and 40,570 new cases of rectal cancer in the 2004 in the United States alone. Colon cancer and rectal cancer will cause about 56,730 deaths combined in the United States.
• ACS Website cancer with the extension .org of the world wide web.
• 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).
• FAP familial adenomatous polyposis
• 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 post- menopausal women. Davila at 50-55. The effect of antioxidants in reducing the risk of colon cancer is unclear. Davila at 53.
• 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.
• 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.
• Davila at 59-60, 61 Davila at 59-60, 61.
• 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.
• stage the cancer 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., Semm. Surg. Oncol. 15(3): 143-150 (1998).
• chest films, liver functionality tests, and liver scans are employed to determine the extent of metastasis. Fleming at 84.
• 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 mmor (T), regional lymph nodes (N), and distant metastasis (M).
• T primary mmor
• N regional lymph nodes
• M distant metastasis
• 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 basement (intramucosal).
• Tis in situ carcinoma
• the cancer has not spread to the regional lymph nodes (NO), and there is no distant metastasis (M0).
• NO regional lymph nodes
• M0 distant metastasis
• stage I there is still no spread of the cancer to the regional lymph nodes and no distant metastasis, but the mmor has invaded the submucosa (TI) or has progressed further to invade the muscularislitis (T2).
• Stage II also involves no spread of the cancer to the regional lymph nodes and no distant metastasis, but the mmor has invaded the subserosa, or the nonperitonealized horric 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).
• stage IV involves any of the T or N substages, as well as distant metastasis. Fleming at 84-85; Burdette at 127.
• 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.
• 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.
• FAP the mmor 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).
• Wntl is a secreted protein gene originally identified within mouse mammary cancers by its insertion into the mouse mammary tumor vims (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.
• 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, Disheveled, which in turn inactivates the glycogen synthesase kinase 3 ⁇ (GSK3 ⁇ ). Taipal (2001). Usually active GSK3 ⁇ will form a complex with the adenomatous polyposis coli (APC) protein and phosphorylate another complex member, ⁇ -catenin.
• APC adenomatous polyposis coli
• ⁇ -catenin is directed to degradation through the ubiquitin pathway.
• GSK3 ⁇ or APC activity is down regulated, ⁇ -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 ⁇ -catenin to Tcf releases the transcriptional repression and induces gene transcription.
• Tcf/Lef lymphocyte excitation factor
• Tcf/Lef T-cell factor or lymphocyte excitation factor
• Binding of ⁇ -catenin to Tcf releases the transcriptional repression and induces gene transcription.
• genes regulated by ⁇ -catenin are a transcriptional repressor Engrailed, a transforming growth factor- ⁇ (TGF- ⁇ ) family member Decapentaplegic, and the cytokine Hedgehog in Drosophila.
• ⁇ -Catenin also involves in regulating cell adhesion by binding to ⁇ -catenin and E-cadherin.
• binding of ⁇ -catenin to these proteins controls the cytoplasmic ⁇ -catenin level and its complexing with TCF. Taipal (2001).
• Growth factor stimulation and activation of c- src or v-src also regulate ⁇ -catenin level by phosphorylation of ⁇ -catenin and its related protein, pl20 cas . When phosphorylated, these proteins decrease their binding to E- cadherin and ⁇ -catenin resulting in the accumulation of cytoplasmic ⁇ -catenin. Reynolds, A.B. et al. Mol. Cell Biol.
• the molecular alternations that occur in this pathway largely involve deletions of alleles of tumor-suppressor genes, such as APC, ⁇ 53 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.
• MMR DNA mismatch repair
• HNPCC Human homologues of the bacterial mutHLS complex (hMSH2, hMLHl, hPMSl, KPMS2 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. Mol. 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).
• TGF ⁇ - RII Fallik, D. et al. Gastroenterol Clin Biol. 24: 917-22 (2000)
• IGFII-R Giovannucci E. J. NMtr. 131: 3109S-20S (2001)
• FAM Focal adhesion kinase
• ECM extracellular matrix
• 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.
• c-src/FAK complexes may coordinately deregulate NEGF expression and apoptosis inhibition.
• a specific signal-transduction pathway for cell survival that implicates integrin engagement leads to FAK activation and thus activates PI-3 kinase and akt.
• 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.
• 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.
• ECM extracellular matrix
• 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.
• Angiogenesis progresses by a stimulus that 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.
• 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.
• a mmor 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 mmor 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 mmor, but also for the dissemination and recolonization of metastatic mmor 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.
• angiogenesis inhibitors 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 mmors can inhibit the growth of distant metastases. O'Reilly and Folkman hypothesized that a primary mmor 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 mmor 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 mmors. It is a proteolytic fragment of a larger protein: endostatin is a 20 kDa fragment of collagen XVIII (amino acid HI 132- K1315 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 mor-bearing mice leads to significant mmor regression, and no toxicity or drug resistance has been observed even after multiple treatment cycles. Boehm et al., 1997, Nature 390(6658):404-407.
• endostatin targets genetically stable endothelial cells and inhibits a variety of solid mmors 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.
• angiogenesis inhibitors have been shown to be more effective when combined with radiation and chemotherapeutic agents. Klement, 2000, J. Clin Invest, 105(8) R15- 24. Browder, 2000, Cancer Res. 6-(7) 1878-86, Arap et al., 1998, Science 279(5349):377- 80; Mauceri et al., 1998, Nature 394(6690):287-91.
• the present invention provides alternative methods of treating ovarian, pancreatic and colon cancer that overcome the limitations of conventional therapeutic methods as well as offer additional advantages that will be apparent from the detailed description below.
• This invention is directed to an isolated Ovrl 15 antibody that binds to Ovrl 15 on a mammalian cell in vivo.
• the invention is further directed to an isolated Ovrl 15 antibody that internalizes upon binding to Ovrl 15 on a mammalian cell in vivo.
• the antibody may be a monoclonal antibody.
• 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 accession number PTA-5202, PTA-5916, PTA-5917, PTA-5918, PTA- 5919 and PTA-5920.
• the invention is further directed to an isolated Ovrl 15 antibody that inhibits Ovrl 15 activity.
• the Ovrl 15 activity is a protease activity.
• 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 accession number PTA-5202, PTA-5916, PTA-5917, PTA-5918, PTA-5919 and PTA-5920.
• 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.
• the anti-Ovrl 15 monoclonal antibody that inhibits the growth of Ovrl 15-expressing cancer cells in vivo.
• the antibody may be produced in bacteria.
• the antibody may be a humanized form of an anti-Ovrl 15 antibody produced by a hybridoma selected from the group of hybridomas having ATCC accession number PTA-5202, PTA-5916, PTA-5917, PTA-5918, PTA-5919 and PTA-5920.
• the cancer is selected from the group consisting of ovarian, pancreatic and colon cancer.
• the invention is also directed to a method of producing the antibodies comprising culmring an appropriate cell and recovering the antibody from the cell culmre.
• 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 Ovrl 15-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, pancreatic and colon cancer cell.
• the ovarian, pancreatic or colon cancer may be ovarian epithelial cancer, pancreatic epithelial cancer or colon epithelial cancer including ovarian serous adenocarcinoma and colon adenocarcinoma or metastatic cancer.
• the invention is also directed to a method of alleviating an Ovrl 15-expressing cancer in a mammal, comprising administering a therapeutically effective amount of the antibodies to the mammal.
• the invention is directed to an article of manufacmre comprising a container and a composition contained therein, wherein the composition comprises an antibody as described herein.
• the article of manufacmre may also comprise an additional component, e.g., a package insert indicating that the composition can be used to treat ovarian, pancreatic and colon cancer.
• FIGURE 1 A shows the results of FACS Analysis of Ovrl 15 Transfected Mouse LMTK Cells.
• FIGURE IB shows the results of FACS analysis Ovrl 15.F76.2 MAb Binds to
• FIGURE 2 shows Ovrl 15 A51.2 binds to the surface of live ovarian CaOV3 cancer cells.
• FIGURE 3 shows Ovrl 15 Cy3-A51.2 binds to live ovarian CaOV3 cancer cells.
• FIGURE 4 shows Ovrl 15 Cy3-A51.2 binds to live ovarian cancer cells and is internalized.
• FIGURE 5 shows Ovrl 15 Cy3-A51.2 binds to live pancreatic cancer cells.
• FIGURE 6 shows the localization of Ovrl 15 in ovarian cancer cells.
• FIGURE 7 shows the localization of Ovrl 15 in ovarian cancer cells.
• FIGURE 8 shows Ovrl 15 D84 labels epithelial cells in ovarian cancer mmors.
• FIGURE 9 shows Ovrl 15 D84 labels epithelial cells in pancreatic cancer mmors.
• FIGURE 10 shows Ovarian Serous Papillary Adenocarcinoma, Unfixed OCT
• FIGURE 11 shows Ovrll5.F64.2 and Ovrll5.D43.1 specifically label frozen and FFPE colon adenocarcinoma cancer cells compared to control Mouse IgGl.
• FIGURE 12 is an epitope map for monoclonal antibodies binding to Ovrl 15.
• FIGURE 13 shows a Sandwich ELISA detection of Ovrl 15 in mmor cell and transfected cell lysates using Ovrl 15.D43 & F-Series MAbs.
• FIGURE 14 shows Anti-Ovrl 15 Activity Screening with Ovrl 15 MAbs.
• FIGURE 15 shows Ovrl 15 ruAbs Recognize Native Ovrl 15 Protein in Human Cell Lines and Ovarian Tumors by Western Blot (mAb Ovrl 15.F21.1).
• FIGURE 16 shows Transformation of Epithelial Cells by Overexpression of
• FIGURE 17 shows Overexpression of Ovrl 15 Induces SQ Tumor Growth in SCID Beige Mice.
• FIGURE 18 shows Expression of Ovrl 15 Protein from Tumor Xenografts.
• FIGURE 19 shows Overexpression of Ovrl 15 Protects RK3E Cells From
• Ovrl 15 has also been disclosed in the REFSEQ database as: NM_019894.2 (GI: 34304348) Homo sapiens transmembrane protease, serine 4 (TMPRSS4), transcript variant 1, mRNA.
• REFSEQ gives the following summary of TMPRSS4 (Ovrl 15):
• This gene encodes a member of the serine protease family. Serine proteases are known to be involved in a variety of biological processes, whose malfunction often leads to human diseases and disorders. This gene was identified as a gene overexpressed in pancreatic carcinoma. The encoded protein is membrane bound with a N-terminal anchor sequence and a glycosylated extracellular region containing the serine protease domain.” The amino acids 52-435 of Ovrl 15 are located on the cell surface. Ovrl 15 as used herein includes allelic variants and conservative substitution mutants of the protein that have Ovrl 15 biological activity.
• TMPRSS4 Homo sapiens transmembrane protease, serine 4
• TMPRSS3 novel transmembrane serine protease
• Wallrapp et al. A novel transmembrane serine protease (TMPRSS3) overexpressed in pancreatic cancer, Cancer Res. 60(10):2602-6 (2000).
• Ovrl 15 is apparently associated to the more aggressive ovarian, pancreatic and colon cancers make this cell surface antigen an attractive target for immunotherapy of these and possibly other mmor types.
• antibody as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g.
• bispecific antibodies bispecific antibodies
• antibody fragments so long as they exhibit the desired biological activity.
• immunoglobulin immunoglobulin
• antibody immunoglobulin
• An “isolated antibody” is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• 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).
• 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 ⁇ , ⁇ and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
• Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
• 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.
• CHI heavy chain
• 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 ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
• the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in C H sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
• 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.
• variability is not evenly distributed across the 1-10-amino acid span of the variable domains.
• 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.
• FRs framework regions
• 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 tliree hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet stracmre.
• 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).
• 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.
• 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.
• each monoclonal antibody is directed against a single determinant on the antigen.
• 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.
• 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.
• PatentNo. 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 that 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.
• the intact antibody has one or more effector functions.
• antibody fragment comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
• 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 (NH), 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.
• Fab' 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 that 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.
• the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired stracmre for antigen binding.
• 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.
• a “native sequence” polypeptide is one that 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.
• amino acid sequence variant refers to a polypeptide that has amino acid sequences that differ to some extent from a native sequence polypeptide.
• amino acid sequence variants of Ovrl 15 will possess at least about 70% homology with the native sequence Ovrl 15, 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, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
• a functional fragment or analog of an antibody is a compound having qualitative biological activity in common with a full-length antibody.
• a functional fragment or analog of an anti-IgE antibody is one that 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, Fc ⁇ RI.
• “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. Mol. 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.
• 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.
• donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• 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.
• 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.
• Fc immunoglobulin constant region
• an anti-Ovrl 15 antibody that "internalizes” is one that is taken up by (i.e., enters) the cell upon binding to Ovrl 15 on a mammalian cell (i.e. cell surface Ovrl 15).
• 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 Ovrl 15-expressing cell, especially an Ovrl 15-expressing cancer cell.
• the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds.
• 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 mmor cell.
• an anti-Ovrl 15 antibody internalizes upon binding Ovrl 15 on a mammalian cell can be determined by various assays including those described in the experimental examples below.
• the test antibody is labeled and introduced into an animal known to have Ovrl 15 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 Ovrl 15-expressing tumor transplant or xenograft, or a mouse into which cells transfected with human Ovrl 15 have been introduced, or a transgenic mouse expressing the human Ovrl 15 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.
• 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.
• the cells can be incubated in tissue culture dishes in the presence or absence of the relevant antibodies added to the culmre media and processed for microscopic analysis at desired time points.
• an internalized, labeled antibody in the cells can be directly visualized by microscopy or by autoradiography if radiolabeled antibody is used.
• a population of cells comprising Ovrl 15-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.
• 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.
• 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.
• 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 kinetics of internalization of the anti-Ovrl 15 antibodies are such that they favor rapid killing of the Ovrl 15-expressing target cell. Therefore, it is desirable that the anti-Ovrl 15 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-Ovrl 15 antibody in vivo.
• the antibody will preferably be internalized into the cell within a few hours upon binding to Ovrl 15 on the cell surface, preferably within 1 hour, even more preferably within 15-30 minutes.
• a cross-blocking assay e.g., a competitive ELISA assay can be performed.
• a competitive ELISA assay Ovrl 15-coated wells of a microtiter plate, or Ovrl 15-coated sepharose beads, are pre-incubated with or without candidate competing antibody and then a biotin-labeled anti-Ovrl 15 antibody of the invention is added.
• the amount of labeled anti-Ovrl 15 antibody bound to the Ovrl 15 antigen in the wells or on the beads is measured using avidin-peroxidase conjugate and appropriate substrate.
• the anti-Ovrl 15 antibody can be labeled, e.g., with a radioactive or fluorescent label or some other detectable and measurable label.
• the amount of labeled anti-Ovrl 15 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-Ovrl 15 antibody of the invention if the candidate competing antibody can block binding of the anti-Ovrl 15 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 Ovrll5.A2.1, Ovrll5.All.l, Ovrll5.A51.2 (also known as Ovrl 15 A51.2), Ovrll5.A63.2, Ovrl l5.D3, Ovrll5.D15, Ovrll5.D20, Ovrll5.D26, Ovrll5.D31, Ovrll5.D32, Ovrll5.D34, Ovrll5.D37, Ovrll5.D43, Ovrl l5.D51, Ovrll5.D69, Ovrll5.D71, Ovrl l5.D81, Ovrll5.D84, Ovrll5.D94, Ov ⁇ ll5.F2, Ovrll5.F3, Ovrll5.F4, Ovrll5.F5, Ovrll5.F6, Ovrll5.F7, Ovrll5.F8, Ovrll5.F9
• Ovrll5.A51.2 (also known as Ovrl 15 A51.2), Ovrll5.A63.2, Ovrll5.D3, Ovrl 15. D 15, Ovrll5.D20, Ovrll5.D26, Ovrll5.D31, Ovrl l5.D32, Ovrll5.D34, Ovrll5.D37, Ovrll5.D43, Ovrll5.D51, Ovrll5.D69, Ovrll5.D71, Ovrll5.D81, Ovrll5.D84, Ovrll5.D94, Ovrl l5.F2, Ovrll5.F3, Ovrll5.F4, Ovrll5.F5, Ovrl l5.F6, Ovrll5.F7, Ovrll5.F8, Ovrll5.F9, Ovrll5.F10, Ovrll5.Fll, Ovrl l5.F13, Ovrll5.F14, Ovrll5.F15,
• Ovrll5.A2.1, Ovrll5.All.l, Ovrll5.A51.2 also known as Ovrl 15 A51.2
• Ovrll5.A63.2 Ovrll5.D3, Ovrll5.D15, Ovrll5.D20, Ovrll5.D26, Ovrll5.D31, Ovrl l5.D32, Ovrl l5.D34, Ovrll5.D37, Ovrll5.D43, Ovrll5.D51, Ovrll5.D69, Ovrll5.D71, Ovrll5.D81, Ovrll5.D84, Ovrll5.D94, Ovrll5.F2, Ovrll5.F3, Ovrll5.F4, Ovrl l5.F5, Ovrll5.F6, Ovrll5.F7, Ovrll5.F8, Ovrll5.F9, Ovrl l5.F10, Ovrll5.F2, Ovrll5.F3,
• an antibody with the biological characteristic of the Ovrl 15.A2.1, Ovrll5.All.l, Ovrl l5.A51.2 also known as Ovrll5 A51.2
• Ovrll5.A63.2 Ovrll5.D3, Ovrl l5.D15, Ovrl l5.D20, Ovrll5.D26, Ovrl l5.D31, Ovrl l5.D32, Ovrll5.D34, Ovrl l5.D37, Ovrl l5.D43, Ovrll5.D51, Ovrll5.D69, Ovrll5.D71, Ovrll5.D81, Ovrll5.D84, Ovrll5.D94, Ovrl l5.F2, Ovrl l5.F3, Ovrll5.F4, Ovrl l5.F5, Ovrll5.F6, Ovrll5.F7, Ovrl l5.F8, Ovrll5.F9
• an antibody with the biological characteristic of the Ovrl 15.A2.1, Ovrll5.All.l, Ovrll5.A51.2 (also known as Ovrl 15 A51.2), Ovrl 15. A63.2, Ovrll5.D3, Ovrll5.D15, Ovrll5.D20, Ovrll5.D26, Ovrll5.D31, Ovrll5.D32, Ovrl l5.D34, Ovrll5.D37, Ovrll5.D43, Ovrll5.D51, Ovrll5.D69, Ovrll5.D71, Ovrl l5.D81, Ovrll5.D84, Ovrll5.D94, Ovrll5.F2, Ovrll5.F3, Ovrll5.F4, Ovrll5.F5, Ovrll5.F6, Ovrll5.F7, Ovrll5.F8, Ovrll5.F9, Ovrll5.F10, Ovrll5.Fll,
• 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 Ovrl 15 protein disclosed herein.
• Methods for identifying antagonists of an Ovrl 15 polypeptide may comprise contacting an Ovrl 15 polypeptide or a cell expressing Ovrl 15 on the cell surface, with a candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the Ovrl 15 polypeptide.
• an “antibody that inhibits the growth of mmor cells expressing Ovrl 15" or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of cancer cells expressing or overexpressing Ovrl 15.
• Preferred growth inhibitory anti-Ovrll5 antibodies inhibit growth of Ovrl 15-expressing tumor cells e.g., ovarian, pancreatic and colon 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 mmor 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 mmor 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-Ovrl 15 antibody at about 1 pg/kg to about 100 mg/kg body weight results in reduction in mmor size or mmor 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 N, fragmentation of D ⁇ A, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
• the cell is usually one that overexpresses Ovrl 15.
• the cell is a mmor cell, e.g. an ovarian, pancreatic and colon cell.
• Various methods are available for evaluating the cellular events associated with apoptosis.
• phosphatidyl serine (PS) translocation can be measured by annexin binding; D ⁇ A fragmentation can be evaluated through D ⁇ A laddering; and nuclear/chromatin condensation along with D ⁇ A fragmentation can be evaluated by any increase in hypodiploid cells.
• 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.
• antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
• ADCC antibody-dependent cell-mediated cytotoxicity
• FcRs Fc receptors
• B cell receptor B cell receptor
• B cell activation B cell activation.
• FcRs Fc receptors
• cytotoxic cells e.g. Natural Killer (NK) cells, neutrophils, and macrophages
• the antibodies “arm" the cytotoxic cells and are absolutely required for such killing.
• NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
• FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
• 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.
• ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an 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.
• a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
• Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
• Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
• Inhibiting receptor Fc ⁇ RIIB 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.
• FcR neonatal receptor
• Human effector cells are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes that 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.
• PBMC peripheral blood mononuclear cells
• NK natural killer cells
• monocytes cytotoxic T cells and neutrophils
• 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 (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen.
• 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.
• cancer examples include, but are not limited to, carcinoma, lymphoma, blastoma, 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
• An “Ovrl 15-expressing cell” is a cell that expresses endogenous or transfected Ovrl 15 on the cell surface.
• a “Ovrl 15-expressing cancer” is a cancer comprising cells that have Ovrl 15 protein present on the cell surface.
• a “Ovrl 15-expressing cancer” produces sufficient levels of Ovrl 15 on the surface of cells thereof, such that an anti- Ovrl 15 antibody can bind thereto and have a therapeutic effect with respect to the cancer.
• a cancer that "overexpresses" Ovrl 15 is one that has significantly higher levels of Ovrl 15 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.
• Ovrl 15 overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the Ovrl 15 protein present on the surface of a cell (e.g. via an immunohistochemistry assay; FACS analysis). Alternatively, or additionally, one may measure levels of Ovrl 15-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).
• FISH fluorescent in situ hybridization
• PCR polymerase chain reaction
• antibody-based assays see also, e.g., U.S. PatentNo. 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)).
• 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.
• An Ovrl 15-expressing cancer includes ovarian, pancreatic, lung or breast 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.
• 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 Ovrl 15-expressing cancer if, after receiving a therapeutic amount of an anti-Ovrl 15 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 mmor 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 mmor metastasis; inhibition, to some extent, of mmor 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.
• the anti-Ovrl 15 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 therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the mmor 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) mmor metastasis; inhibit, to some extent, mmor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See preceding definition of "treating".
• 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.
• 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.
• physiologically acceptable carrier is an aqueous pH buffered solution.
• 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 TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
• buffers such as phosphate, citrate, and other organic acids
• antioxidants including ascorbic acid
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destraction of cells.
• the term is intended to include radioactive isotopes (e.g. At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , and radioactive isotopes of Lu), chemotherapeutic agents e.g.
• methotrexate adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorabicin, 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 mmor cells.
• a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially an Ovrl 15-expressing cancer cell, either in vitro or in vivo.
• the growth inhibitory agent may be one which significantly reduces the percentage of Ovrl 15-expressing cells in S phase.
• 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 doxorabicin, epirubicin, daunorubicin, etoposide, and bleomycin.
• 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 drags" by Murakami et al. (WB Saunders:
• the taxanes 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 micrombules from mbulin dimers and stabilize micrombules by preventing depolymerization, which results in the inhibition of mitosis in cells.
• Label refers to a detectable compound or composition that 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 that is detectable.
• epitope tagged refers to a chimeric polypeptide comprising an anti-Ovrl 15 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
• package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, admimstration, contraindications and/or warnings concerning the use of such therapeutic products.
• 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 that 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.
• a plasmid construct will also include an origin of replication (e.g., the ColEl origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication 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-Ovrl 15 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.
• dsRNA double stranded RNAs
• 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 mRNA by ribonuclease L.
• dsRNA short interfering RNAs
• dicer 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, Namre, 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 stracmre 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).
• RISC RNA-induced silencing complex
• RNAi mediated RNAi Short interfering RNA mediated RNAi has been studied in a variety of systems. Fire et al., 1998, Namre, 391, 806, were the first to observe RNAi in C. Elegans. Wianny and Goetz, 1999, Namre Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Namre, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Namre, 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.
• RNAi activity Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity.
• these smdies 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 smdies have indicated that a 5'-phos ⁇ hate 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).
• 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'-0-methyl nucleotides, and nucleotides containing a 2 -0 or 4'-C methylene bridge.
• PKR double stranded-RNA-dependent protein kinase
• 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.
• 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
• 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.
• the invention provides anti-Ovrl 15 antibodies.
• the anti-Ovrl 15 antibodies internalize upon binding to cell surface Ovrl 15 on a mammalian cell.
• the anti- Ovrl 15 antibodies may also destroy or lead to the destraction of mmor cells bearing Ovrl 15.
• Ovrl 15 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 Ovrl 15 is internalization competent upon binding by the anti- Ovrl 15 antibodies of the invention. Additionally, it was demonstrated that the anti- Ovrl 15 antibodies of the present invention can specifically target Ovrl 15-expressing mmor cells in vivo and inhibit or kill these cells.
• the anti-Ovrl 15 antibodies make these antibodies very suitable for therapeutic uses, e.g., in the treatment of various cancers including ovarian, pancreatic and colon cancer.
• Internalization of the anti-Ovrl 15 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 mmor cell by ADCC or some other mechanism.
• the anti-Ovrl 15 antibodies of the invention also have various non-therapeutic applications.
• the anti-Ovrl 15 antibodies of the present invention can be useful for diagnosis and staging of Ovrl 15-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 Ovrl 15 from cells, for detection and quantisation of Ovrl 15 in vitro, e.g. in an ELISA or a Western blot, to kill and eliminate Ovrl 15-expressing cells from a population of mixed cells as a step in the purification of other cells.
• the internalizing anti-Ovrl 15 antibodies of the invention can be in the different forms encompassed by the definition of "antibody” herein.
• 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.
• 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.
• 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.
• ADCC antibody-dependent cellular cytotoxicity
• 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-Ovrl 15 antibodies of the invention are also contemplated, e.g., an anti- Ovrl 15 antibody which has the biological characteristics of a monoclonal antibody produced by the hybridomas accorded ATCC accession numbers PTA-5202, PTA-5916, PTA-5917, PTA-5918, PTA-5919 and PTA-5920, specifically including the in vivo mmor targeting, internalization and any cell proliferation inhibition or cytotoxic characteristics.
• anti-Ovrl 15 antibodies that bind to an epitope present in amino acids 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180,180-190, 190-200, 200-210, 210-220, 220-230, 230-240, 240- 250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430- 435 of human Ovrl 15.
• the present anti-Ovrl 15 antibodies are useful for treating an Ovrl 15-expressing cancer or alleviating one or more symptoms of the cancer in a mammal.
• a cancer includes ovarian, pancreatic and colon cancer, cancer of the urinary tract, lung cancer, breast cancer and prostate cancer.
• Such a cancer includes more specifically, ovarian serous adenocarcinoma, breast infiltrating ductal carcinoma, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma.
• the breast cancer may be HER-2 negative or positive breast cancer.
• the cancers encompass metastatic cancers of any of the preceding, e.g., ovarian, pancreatic and colon cancer metastases.
• the antibody is able to bind to at least a portion of the cancer cells that express Ovrl 15 in the mammal and preferably is one that does not induce or that minimizes HAMA response.
• the antibody is effective to destroy or kill Ovrl 15-expressing mmor cells or inhibit the growth of such mmor cells, in vitro or in vivo, upon binding to Ovrl 15 on the cell.
• Such an antibody includes a naked anti-Ovrl 15 antibody (not conjugated to any agent). Naked anti-Ovrl 15 antibodies having mmor 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 mmor cell destruction.
• Cytotoxic properties can be conferred to an anti-Ovrl 15 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-Ovrl 15 antibody of the invention, and a carrier.
• compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-Ovrl 15 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- Ovrl 15 antibody of the invention, and a carrier.
• the formulation may be a therapeutic formulation comprising a pharmaceutically acceptable carrier.
• nucleic acids encoding the internalizing anti-Ovrl 15 antibodies are encompassed.
• the invention also provides methods useful for treating an Ovrl 15-expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an internalizing anti-Ovrl 15 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 Ovrl 15 expressing cell.
• kits and articles of manufacmre comprising at least one antibody of this invention, preferably at least one anti-Ovrl 15 antibody of this invention that binds to Ovrl 15 on a mammalian cell in vivo or at least one internalizing anti-Ovrl 15 antibody of this invention.
• Kits containing anti-Ovrl 15 antibodies find use in detecting Ovrl 15 expression, or in therapeutic or diagnostic assays, e.g., for Ovrl 15 cell killing assays or for purification and/or immunoprecipitation of Ovrl 15 from cells.
• kits for isolation and purification of Ovrl 15, can contain an anti-Ovrl 15 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 Ovrl 15 in vitro, e.g. in an
• Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.
• the Ovrl 15 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 Ovrl 15 lacking the membrane spanning sequence, or synthetic peptides to selected portions of the protein.
• cells expressing Ovrl 15 at their cell surface e.g. CHO or NIH-3T3 cells transformed to overexpress Ovrl 15; ovarian, pancreatic, lung, breast or other
• Ovrl 15-expressing mmor cell line or membranes prepared from such cells can be used to generate antibodies.
• the nucleotide and amino acid sequences of human and murine Ovrl 15 are available as provided above.
• Ovrl 15 can be produced recombinantly in and isolated from, prokaryotic cells, e.g., bacterial cells, or eukaryotic cells using standard recombinant DNA methodology.
• Ovrl 15 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 Ovrl 15 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., Mol. Cell.
• Tag polypeptides include the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an a-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 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.
• sc subcutaneous
• ip intraperitoneal
• Conjugates also can be made in recombinant cell culmre 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 infradermally at multiple sites.
• 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.
• aggregating agents such as alum are suitably used to enhance the immune response.
• Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Namre, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567).
• 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.
• lymphocytes may be immunized in vitro.
• 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)).
• a fusion partner e.g., a myeloma cell line using a suitable fusing agent, such as polyethylene glycol
• the hybridoma cells thus prepared are seeded and grown in a suitable culmre 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.
• a suitable culmre 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.
• the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
• HGPRT hypoxanthine guanine phosphoribosyl transferase
• 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- 11 mouse mmors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culmre 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.
• Culmre medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
• 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).
• 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 culmre media for this purpose include, for example, D-MEM or RPMI-1640 medium.
• the hybridoma cells may be grown in vivo as ascites mmors 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 culmre 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, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.
• affinity chromatography e.g., using protein A or protein G-Sepharose
• ion-exchange chromatography e.g., hydroxylapatite chromatography
• gel electrophoresis e.g., 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.
• 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.
• 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
• the DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substimting human heavy chain and light chain constant domain (CH and CL) sequences for the homologous murine sequences (U.S. PatentNo. 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 sequence for a non-immunoglobulin polypeptide (heterologous polypeptide).
• CH and CL human heavy chain and light chain constant domain
• nonimmunoglobulin polypeptide sequences can substimte 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.
• 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., Namre, 321:522-525 (1986); Reichmann et al., Namre, 332:323- 327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substimting hypervariable region sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. PatentNo. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• 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.
• variable domains both light and heavy
• HAMA response human anti-mouse antibody
• the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences.
• the human N 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. Mol. 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. ⁇ atl. 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.
• 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.
• 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.
• the humanized antibody may be an intact antibody, such as an intact IgGl antibody.
• human antibodies can be generated.
• transgenic animals e.g., mice
• transgenic animals e.g., mice
• J H antibody heavy-chain joining region
• 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.
• phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
• 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.
• a filamentous bacteriophage such as M13 or fd
• selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
• 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).
• V-gene segments can be used for phage display.
• Clackson et al., Namre, 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. Mol. 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.
• 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 mmors.
• 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 culmre. 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).
• scFv single chain Fv fragment
• 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 are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Ovrl 15 protein. Other such antibodies may combine an Ovrl 15 binding site with a binding site for another protein. Alternatively, an anti-Ovrl 15. Arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a Tcell receptor molecule (e.g. C133), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16), so as to focus and localize cellular defense mechanisms to the Ovrl 15-expressing cell.
• a triggering molecule such as a Tcell receptor molecule (e.g. C133), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16), so
• Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Ovrl 15. These antibodies possess an Ovrl 15-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon- ⁇ , 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-Fc ⁇ RIII antibody and U.S. PatentNo. 5,837,234 discloses a bispecific anti-ErbB2/anti-Fc ⁇ RI antibody. A bispecific anti-ErbB2/Fc ⁇ antibody is shown in WO98/02463.
• U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.
• 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 et al, 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 stracmre. 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).
• antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
• the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C H 2, and C H 3 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.
• 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 stracmre 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 bispecific 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.
• the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culmre.
• the preferred interface comprises at least a part of the CH3 domain.
• 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.
• 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. PatentNo. 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.
• 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.
• bispecific antibodies have been produced using leucine zippers.
• 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 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 Graber et al., J. Immunol., 152:5368 (1994).
• Antibodies with more than two valencies are contemplated.
• trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
• 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. tefravalent 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.
• the polypeptide chain(s) may comprise VDl(Xln-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.
• 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.
• Amino acid sequence modification(s) of the anti-Ovrl 15 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-Ovrl 15 antibody are prepared by introducing appropriate nucleotide changes into the anti-Ovrl 15 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-Ovrl 15 antibody. Any combination of deletion, insertion, and substimtion 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-Ovrl 15 antibody, such as changing the number or position of glycosylation sites.
• a useful method for identification of certain residues or regions of the anti-Ovrl 15 antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis” as described by Cunningham and Wells in Science, 244:1081-1085 (1989).
• a residue or group of target residues within the anti-Ovrl 15 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 Ovrl 15 antigen.
• charged residues such as arg, asp, his, lys, and glu
• a neutral or negatively charged amino acid most preferably alanine or polyalanine
• 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.
• the site for introducing an amino acid sequence variation is predetermined, the namre of the mutation per se need not be predetermined.
• ala scanning or random mutagenesis is conducted at a target codon or region and the expressed anti-Ovrl 15 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 acid residues.
• terminal insertions include an anti-Ovrl 15 antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
• Other insertional variants of the anti- Ovrl 15 antibody molecule include the fusion to the N- or C-terminus of the anti-Ovrl 15 antibody to an enzyme (e.g. for ADEPT) or a fusion to a polypeptide which increases the serum half-life of the antibody.
• variants are an amino acid substimtion variant. These variants have at least one amino acid residue in the anti-Ovrl 15 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 substimtions are shown in Table I under the heading of "preferred substimtions”. If such substimtions result in a change in biological activity, then more substantial changes, denominated "exemplary substimtions" 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.
• Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the stracmre 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 substimtions 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-Ovrl 15 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
• 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 substimting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
• a parent antibody e.g. a humanized or human antibody
• 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 substimtions 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.
• alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
• Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
• 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.
• 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-Ovrl 15 antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a namral source (in the case of namrally 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-Ovrl 15 antibody. It may be desirable to modify the antibody of the invention with respect to effector function, e.g.
• ADCC antigen-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• This may be achieved by introducing one or more amino acid substimtions in an Fc region of the antibody.
• 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).
• 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).
• a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example.
• the term "salvage receptor binding epitope” refers to an epitope of the Fc region of the antibody.
• Desired properties of an anti-Ovrl 15 antibody include cell growth inhibitory effects, mmor growth inhibitory effects, cell proliferation inhibitory effects, mmor proliferation inhibitory effects, cell killing effects, mmor killing effects, cytostatic effects on mmors, protein function inhibitory effects and removal of protein from cell surface.
• a highly desired property of an anti-Ovrl 15 antibody is inhibition of Ovrl 15 protease activity.
• an anti-Ovrl 15 antibody of the invention listed above may be assessed by methods known in the art, e.g., using cells which express Ovrl 15 either endogenously or following transfection with the Ovrl 15 gene.
• the mmor cell lines and Ovrl 15-transfected cells provided in Example 1 below may be treated with an anti-Ovrl 15 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 3 H-thymidine uptake by the cells treated in the presence or absence an anti-Ovrl 15 antibody of the invention.
• 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 mmor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below.
• the mmor cell is one that over-expresses Ovrl 15.
• the anti-Ovrl 15 antibody will inhibit cell proliferation of an Ovrl 15-expressing mmor cell in vitro or in vivo by about 25-100% compared to the untreated mmor 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 ⁇ g/ml.
• Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 ⁇ g/ml or about 0.5 nM to 200nM in cell culmre, where the growth inhibition is determined 1-10 days after exposure of the mmor cells to the antibody.
• the antibody is growth inhibitory in vivo if administration of the anti-Ovrl 15 antibody at about l ⁇ g/kg to about lOOmg/kg body weight results in reduction in mmor size or mmor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
• PI uptake assay can be performed in the absence of complement and immune effector cells.
• Ovrl 15-expressing mmor cells are incubated with medium alone or medium containing of the appropriate monoclonal antibody at e.g., about lO ⁇ g/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 (1ml per tube, 3 tubes per treatment group) for removal of cell clumps.
• Tubes then receive PI (lO ⁇ g/ml). Samples may be analyzed using a FACSCANTM flow cytometer and FACSCONNERTTM 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.
• 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-Ovrl 15 antibody of the invention.
• epitope mapping can be performed by methods known in the art.
• 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.
• peptides corresponding to different regions of Ovrl 15 can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.
• a method to screen for antibodies that bind to an epitope which is bound by an antibody this invention may comprise combining an Ovrl 15-containing sample with a test antibody and an antibody of this invention to form a mixture , the level of Ovrl 15 antibody bound to Ovrl 15 in the mixture is then determined and compared to the level of Ovrl 15 antibody bound in the mixture to a control mixture, wherein the level of Ovrl 15 antibody binding to Ovrl 15 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-Ovrl 15 antibody of this invention.
• the level of Ovrl 15 antibody bound to Ovrl 15 is determined by ELISA.
• the control may be a positive or negative control or both.
• the control may be a mixture of Ovrl 15, Ovrl 15 antibody of this invention and an antibody known to bind the epitope bound by the Ovrl 15 antibody of this invention.
• the Ovrl 15 may be bound to a solid support, e.g., a tissue culture plate or to beads, e.g., sepharose beads.
• 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.
• an anti-cancer agent such as a cytotoxic agent or a growth inhibitory agent.
• Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein. Maytansine and maytansinoids
• an anti-Ovrl 15 antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.
• Maytansinoids are mitototic inhibitors which act by inhibiting mbulin 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. PatentNo. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent Nos.
• maytansine and maytansinoids have been conjugated to antibodies specifically binding to mmor 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 culmred colon cancer cells, and showed antitumor activity in an in vivo mmor 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 drag 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-Ovrl 15 antibody-maytansinoid conjugates are prepared by chemically linking an anti-Ovrl 15 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 namral sources. Suitable maytansinoids are disclosed, for example, in U.S. PatentNo.
• Prefened maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
• 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 0 425 235 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 prefened.
• 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-
• Particularly prefened 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.
• SPDP N-succinimidyl (2-pyridyldithio) propionate
• SPP N-succinimidyl (2-pyridylthio)pentanoate
• the linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link.
• 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.
• the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.
• Another immunoconjugate of interest comprises an anti-Ovrl 15 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, ⁇ , ⁇ 2 ⁇ , & ⁇ , N-acetyl- y x PSAG and ⁇ i 1 , (Hin an 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 drag that the antibody can be conjugated is QFA which is an antifolate.
• QFA 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 include, but are not limited to, ⁇ , ⁇ 2 ⁇ , & ⁇ , N-acetyl- y x PSAG and ⁇
• 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, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
• 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).
• a compound with nucleolytic activity e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase.
• the antibody may comprise a highly radioactive atom.
• a variety of radioactive isotopes are available for the production of radioconjugated anti-Ovrl 15 antibodies. Examples include At 211 , 1 131 , 1 125 , In m ,Y 90 , Re , Re , Sm , Bi , P , and radioactive isotopes of Lu.
• the conjugate When used for diagnosis, it may comprise a radioactive atom for scintigraphic smdies, for example Tc 99M or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-Ill, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
• NMR nuclear magnetic resonance
• the radio- or other labels may be incorporated in the conjugate in known ways.
• 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 , I 123 , In 111 , Re 186 , Re 188 , can be attached via a cysteine residue in the peptide.
• Yttrium-90 can be attached via a lysine residue.
• 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-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 bis (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-difluor
• 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 drag in the cell.
• an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992); U.S.
• Patent No. 5,208,020 may be used.
• a fusion protein comprising the anti-Ovrl 15 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.
• 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).
• a "receptor” such streptavidin
• a ligand e.g. avidin
• cytotoxic agent e.g. a radionucleotide
• ADPT Antibody Dependent Enzyme Mediated Prodrag Therapy
• the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrag-activating enzyme which converts a prodrag (e.g. a peptidyl chemotherapeutic agent, see W081/01145) to an active anti-cancer drug.
• a prodrag e.g. a peptidyl chemotherapeutic agent, see W081/01145
• W081/01145 a prodrag-activating enzyme which converts a prodrag
• a prodrag e.g. a peptidyl chemotherapeutic agent, see W081/01145
• 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 prodrags into free drags; arylsulfatase useful for converting sulfate-containing prodrags into free drags; cytosine deaminase useful for converting non-toxic fluorocytosine into the anticancer drug, 5-fluorouracil; proteases, such as senatia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrags into free drugs; D-alanylcarboxypeptidases, useful for converting prodrags that contain D-amin
• antibodies with enzymatic activity can be used to convert the prodrags of the invention into free active drugs (see, e.g., Massey, Namre 328: 457-458 (1987)).
• Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a mmor cell population.
• the enzymes of this invention can be covalently bound to the anti-Ovrl 15 antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
• 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., Namre, 312: 604-608 (1984).
• 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 poly(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• the anti-Ovrl 15 antibodies disclosed herein may also be formulated as immunoliposomes.
• a "liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drag to a mammal.
• the components of the liposome are commonly ananged in a bilayer formation, similar to the lipid anangement 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.
• 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).
• the invention also provides isolated nucleic acid molecule encoding the humanized anti-Ovrl 15 antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the 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.
• the anti-Ovrl 15 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 mamre protein or polypeptide.
• a heterologous polypeptide which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N- terminus of the mamre 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.
• 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.
• a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
• 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.
• 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-Ovrl 15 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.
• 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.
• origins of replication or autonomously replicating 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 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenoviras, VSV or BPN) are useful for cloning vectors in mammalian cells.
• the origin of replication component is not needed for mammalian expression vectors (the SN40 origin may typically be used only because it contains the early promoter).
• Selection genes 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 drag to anest 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.
• Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-Ovrl 15 antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -11, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
• DHFR thymidine kinase
• metallothionein-I and -11 preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
• 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.
• Mtx methotrexate
• DHFR Chinese hamster ovary
• ATCC CRL- 9096 Chinese hamster ovary
• host cells transformed or co-transformed with DNA sequences encoding anti-Ovrl 15 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., Namre, 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 trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
• vectors derived from the 1.6 pm circular plasmid pKDI can be used for transformation of Kluyveromyces yeasts.
• 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 mamre recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
• Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-Ovrl 15 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 (tip) promoter system, and hybrid promoters such as the tac promoter.
• 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-Ovrl 15 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.
• suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde phosphate dehydrogenase, hexokinase, pyravate decarboxylase, phosphofructokinase, glucose phosphate isomerase, 3-phosphoglycerate mutase, pyravate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
• 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde phosphate dehydrogenase, hexokinase, pyravate decarboxylase, phosphofructokinase, glucose phosphate isomerase, 3-phosphoglycerate mutase, pyravate kinase
• 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-Ovrl 15 antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma viras, fowlpox viras, adenoviras (such as Adenoviras 2), bovine papilloma virus, avian sarcoma virus, cytomegaloviras, a retroviras, hepatitis-B viras and most preferably Simian Viras 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.
• viruses such as polyoma viras, fowlpox viras, adenoviras (such as Adenoviras 2), bovine papilloma virus, avian sarcoma
• 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 cytomegaloviras is conveniently obtained as a Hindlll E restriction fragment.
• a system for expressing DNA in mammalian hosts using the bovine papilloma viras as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. PatentNo. 4,601,978.
• Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell viras. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegaloviras early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviras enhancers.
• the enhancer may be spliced into the vector at a position 5 ' or 3' to the anti- Ovrl 15 antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
• Expression vectors used in eukaryotic host cells 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-Ovrl 15 antibody.
• One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vector disclosed therein.
• 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, Senatia, e.g., Senatia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
• E. coli 294 ATCC 31,446
• E. coli B E. coli XI 776
• E. coli W31 10 ATCC 27,325
• 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.
• a cytotoxic agent e.g., a toxin
• 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.
• eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-Ovrl 15 antibody-encoding vectors.
• Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
• 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 K. marxianus
• yanowia EP 402,226
• Pichia pastoris EP 183,070
• Candida Trichoderma reesia
• Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
• 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-Ovrl 15 antibody are derived from multicellular organisms.
• invertebrate cells include plant and insect cells.
• Numerous baculoviral strains and variants and conesponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fraitfly), and Bombyx mori have been identified.
• a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPN and the Bm-5 strain of Bombyx mori ⁇ PN, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera fragiperda cells.
• Plant cell cultures of cotton, corn, 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 culmre 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 Mol Biol 32: 979-986.
• culmre tissue culmre
• useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culmre, 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.
• mice 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 (HELA, 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 mmor (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 transfonned with the above-described expression or cloning vectors for anti-Ovrl 15 antibody production and culmred 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-Ovrl 15 antibody of this invention may be culmred 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 culmring 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 GENTAMYCINTM drag), 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 culmre 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.
• the 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, supematants 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 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 prefened purification technique.
• affinity chromatography being the prefened 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 ⁇ l, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
• Protein G is recommended for all mouse isotypes and for human ⁇ 3 (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.
• the antibody comprises a CH3 domain
• the Bakerbond ABXTMresin J. T. Baker, Philhpsburg, NJ
• 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).
• compositions 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, asparag
• 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.
• an additional antibody e.g.
• the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant.
• 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 drag delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
• colloidal drag delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• 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.
• the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
• the anti-Ovrl 15 antibody that internalizes upon binding Ovrl 15 on a cell surface is used to treat a subject in need thereof having a cancer characterized by Ovrl 15-expressing cancer cells, in particular, ovarian, pancreatic, lung or breast cancer, such as ovarian serous adenocarcinoma or breast infiltrating ductal carcinoma cancer, and associated metastases.
• a cancer characterized by Ovrl 15-expressing cancer cells in particular, ovarian, pancreatic, lung or breast cancer, such as ovarian serous adenocarcinoma or breast infiltrating ductal carcinoma cancer, and associated metastases.
• the cancer will generally comprise Ovrl 15-expressing cells, such that the anti- Ovrl 15 antibody is able to bind thereto. While the cancer may be characterized by overexpression of the Ovrl 15 molecule, the present application further provides a method for treating cancer which is not considered to be an Ovrl 15-overexpressing cancer. This invention also relates to methods for detecting cells which overexpress Ovrl 15 and to diagnostic kits useful in detecting cells expressing Ovrl 15 or in detecting Ovrl 15 in serum from a patient.
• 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 Ovrl 15 overexpressing cells. A level of Ovrl 15 binding higher than that of such a control sample would be indicative of the test sample containing cells that overexpress Ovrl 15.
• the control may be a sample of cells known to contain cells that overexpress Ovrl 15. In such a case, a level of Ovrl 15 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 Ovrl 15.
• Ovrl 15 overexpression may be detected with a various diagnostic assays.
• over expression of Ovrl 15 may be assayed by immunohistochemistry (IHC).
• IHC immunohistochemistry
• Panafin embedded tissue sections from a mmor biopsy may be subjected to the IHC assay and accorded an Ovrl 15 protein staining intensity criteria as follows. Score 0 no staining is observed or membrane staining is observed in less than 10% of mmor cells.
• Score 1+ a faint/barely perceptible membrane staining is detected in more than 10% of the mmor 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
• FISH assays such as the INFORMTM (sold by Ventana, Arizona) or PATHVISIONTM (VySiS, Illinois) may be canied out on formalin- fixed, paraffin-embedded mmor tissue to determine the extent (if any) of Ovrl 15 overexpression in the mmor.
• Ovrl 15 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 Ovrl 15 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 detectable label e.g. a radioactive isotope or a fluorescent label
• a sample suspected of containing cells expressing or overexpressing Ovrl 15 is combined with the antibodies of this invention under conditions suitable for the specific binding of the antibodies to Ovrl 15. Binding and/or internalizing the Ovrl 15 antibodies of this invention is indicative of the cells expressing Ovrl 15.
• the level of binding may be determined and compared to a suitable control, wherein an elevated level of bound Ovrl 15 as compared to the control is indicative of Ovrl 15 overexpression.
• the sample suspected of containing cells overexpressing Ovrl 15 may be a cancer cell sample, particularly a sample of an ovarian cancer, e.g. ovarian serous adenocarcinoma, or a breast cancer, e.g., a breast infiltrating ductal carcinoma.
• a serum sample from a subject may also be assayed for levels of Ovrl 15 by combining a serum sample from a subject with an Ovrl 15 antibody of this invention, determining the level of Ovrl 15 bound to the antibody and comparing the level to a control, wherein an elevated level of Ovrl 15 in the serum of the patient as compared to a control is indicative of overexpression of Ovrl 15 by cells in the patient.
• the subject may have a cancer such as e.g., an ovarian cancer, e.g. ovarian serous adenocarcinoma, or a breast cancer, e.g., a breast infiltrating ductal carcinoma.
• ovarian, pancreatic, lung or breast 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-Ovrl 15 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 mmor targeting and internalizing anti-Ovrl 15 antibodies of the invention are useful to alleviate Ovrl 15-expressing cancers, e.g., ovarian, pancreatic, lung or breast cancers upon initial diagnosis of the disease or during relapse.
• the anti-Ovrl 15 antibody can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy, notably for ovarian, pancreatic, lung or breast cancers, also particularly where shed cells cannot be reached.
• Anti-Ovrl 15 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® (palictaxel), estramustine and mitoxantrone are used in treating metastatic and hormone refractory ovarian, pancreatic, lung or breast cancer, in particular, in good risk patients.
• the cancer patient can be administered anti-Ovrl 15 antibody in conjunction with treatment with the one or more of the preceding chemotherapeutic agents.
• combination therapy with palictaxel and modified derivatives is contemplated.
• the anti-Ovrl 15 antibody will be administered with a therapeutically effective dose of the chemotherapeutic agent.
• the anti-Ovrl 15 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 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.
• an immunoconjugate comprising the anti-Ovrl 15 antibody conjugated with a cytotoxic agent may be administered to the patient.
• the immunoconjugate bound to the Ovrl 15 protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds.
• 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-Ovrl 15 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, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
• the antibodies or immunoconjugates may be injected directly into the mmor mass. Intravenous or subcutaneous administration of the antibody is prefened. Other therapeutic regimens may be combined with the administration of the anti-Ovrl 15 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.
• 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 mmor antigen associated with the Ovrl 15-expressing tumor cells.
• the cocktail may also comprise antibodies that are directed to other epitopes of Ovrl 15. 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-Ovrl 15 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.
• 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
• 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
• 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-Ovrl 15 antibody (and optional
• a cardioprotectant to prevent or reduce myocardial dysfunction associated with the therapy
• one or more cytokines to the patient.
• 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 co- administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-Ovrl 15 antibody.
• 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.
• the antibody is administered by intravenous infusion or by subcutaneous injections.
• 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-Ovrl 15 antibody.
• 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.
• 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.
• 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
• nucleic acid molecule is injected directly into the patient, usually at the site where the antibody is required.
• 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).
• porous membranes which are implanted into the patient.
• nucleic acid is transfened into culmred 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 cunently prefened in vivo nucleic acid molecule transfer techniques include transfection with viral vectors (such as adenoviras, Herpes simplex I viras, 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).
• viral vectors such as adenoviras, Herpes simplex I viras, or adeno- associated virus
• lipid-based systems useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example.
• the invention also relates to an article of manufacmre containing materials useful for the detection for Ovrl 15 overexpressing cells and/or the treatment of Ovrl 15 expressing cancer, in particular ovarian, pancreatic and colon 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 manufacmre 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 Ovrl 15 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- Ovrl 15 antibody of the invention.
• the label or package insert indicates that the composition is used for detecting Ovrl 15 expressing cells and/or for treating ovarian, pancreatic and colon cancer, or more specifically ovarian serous adenocarcinoma, breast infiltrating ductal carcinoma, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma, in a patient in need thereof.
• the breast cancer may be HER-2 negative or positive breast cancer.
• the cancers encompass metastatic cancers of any of the preceding, e.g., ovarian, pancreatic and colon cancer metastases.
• the label or package insert may further comprise instructions for administering the antibody composition to a cancer patient.
• the article of manufacmre 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.
• BWFI bacteriostatic water for injection
• phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
• BWFI bacteriostatic water for injection
• the article of manufacmre may further include other materials desirable from a commercial
• Kits are also provided that are useful for various purposes , e.g., for Ovrl 15 cell killing assays, for purification or immunoprecipitation of Ovrl 15 from cells or for detecting the presence of Ovrl 15 in a serum sample or detecting the presence of Ovrl 15- expressing cells in a cell sample.
• the kit can contain an anti-Ovrl 15 antibody coupled to a solid support, e.g., a tissue culmre plate or beads (e.g., sepharose beads). Kits can be provided which contain the antibodies for detection and quantitation of Ovrl 15 in vitro, e.g. in an ELISA or a Western blot.
• 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.
• Ovrll5.F4 Ovrll5.F5, Ovrll5.F6, Ovrll5.F7, Ovrll5.F8, Ovrll5.F9, Ovrll5.F10, Ovrl l5.Fll, Ovrll5.F13, Ovrll5.F14, Ovrll5.F15, Ovrll5.F19, Ovrll5.F20, Ovrll5.F21, Ovrll5.F22, Ovrll5.F23, Ovrll5.F24, Ovrll5.F25, Ovrll5.F26, Ovrll5.F28, Ovrll5.F29, Ovrll5.F30, Ovrll5.F31, Ovrll5.F32, Ovrll5.F33, Ovrl l5.F34, Ovrll5.F35, Ovrll5.F36, Ovrll5.F37, Ovrll5.F38, Ovrll5.F39, Ovrll5.F
• Ovrl 15.F81, Ovrl 15.F82 and Ovrl 15.F83 If the MAb has been cloned, it will get the nomenclature "X.l,” e.g., the first clone of A7 will be refened to as A7.1, the second clone of A7 will be refened to as A7.2, etc.
• a reference to A7 will include all clones, e.g., A7.1, A7.2, etc. Immunogens and Antigens (Recombinant Proteins. HA & His Tags & Transfected Cells)
• Ovrl 15 (TMPRSS4) constract encoding a tobacco etch virus protease (TEV) recognition site, the serine protease domain of Ovrl 15 from Val203 to Leu435 was cloned in-frame to the C-terminus of glutathione S-transferase (GST) so that the Ovrl 15 constract was expressed as a GST-fusion protein of 486 amino acids using standard techniques.
• Ovrl 15 Purification of Ovrl 15 was completed via glutathione sepharose column.
• Ovrl 15 (TMPRSS4) constract encoding a region of Ovrl 15 from Lys52 to Leu435 was cloned with a six-histidine tag immediately downstream of codon Leu435 and expressed in insect cells using standard baculoviras techniques for production of the D-series MAbs.
• this protein was cloned into a standard expression vector and expressed in mammalian cells using standard technology.Ovrl 15 was purified using Ni-NTA resin.
• Ovrl 15 extracellular construct (Ovrl 15 Lys52-Leu435) amino acid sequence (6 His tag sequence is in bold type) (SEQ ID NO.2)
• HA-tagged Ovrl 15 (Metl-Leu435) was transfected into mouse LMTK cells after cloning into a mammalian vector with an HA tag. Individual clones were checked for expression of Ovrl 15 by western blot using anti-HA antibody (Covance, Richmond, CA), after 1 week in culmre. Ovrl 15 transfected LMTK and 293F amino acid sequence (SEQ ID NO.3)
• Pro 104 was used to screen out cross reactive hybridoma clones, since this antigen was also upregulated in ovarian and pancreatic cancers and since it also contained a potentially cross-reactive serine protease domain.
• Pro 104 (Ile42-Trp297) expressed amino acid sequence (underlined portion represents the honey bee melletin secretion signal and the bold type represents the hexa histidine tag) (SEQ ID NO. 4)
• Pro 104 (Testisin) LMTK Cell Expressed Sequences & Protein Production
• a full length constract encoding Pro 104 (Metl-Nal314) with an HA tag codons located at the C-terminus, downstream from the recombination site was expressed using standard mammalian expression techniques.
• mice were immunized with expressed soluble Ovrl 15 recombinant protein, which conesponded to the serine protease domain (Val203 to Leu435) of the native protein.
• Groups of 8 BALB/c mice were immunized intradermally in both rear footpads. All injections were 25 uL per foot.
• the first injection (day 1) 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, Saint Louis, MS).
• DPBS Dulbecco's phosphate buffered saline
• mice were immunized as above with soluble insect expressed or mammalian expressed Ovrl 15 recombinant protein (respectively). Both of these proteins conesponded to the entire extracellular domain (Lys52-Leu435) of the native Ovrl 15 protein, in order to generate MAbs of both in- vivo therapeutic and diagnostic utility.
• lymph node popliteal tissue
• Lymph node cells were dispersed by pressing through a sterile sieve into DMEM and removing T-cells via anti- CD90 (Thyl.2) coated magnetic beads (Miltenyl Biotech, Baraisch-Gladbach, Germany).
• Monoclonal cultures consisting of the genetically uniform progeny from single cells, were established after the screening procedure above, by sorting of single viable cells into wells of two 96 well plates, using flow cytometry (Coulter Elite).
• the resulting murine B-cell hybridoma cultures were expanded using standard tissue culmre techniques. Selected hybridomas were cryopreserved in fetal bovine serum (FBS) with 10% DMSO and stored in Liquid Nitrogen at -196°C to assure maintenance of viable clone cultures.
• FBS fetal bovine serum
• Hybridoma cell lines were selected for production of Ovrl 15 specific antibody by enzyme linked solid phase immunoassay (ELISA).
• Ovrl 15 or Prol04 proteins were nonspecifically adsorbed to wells of 96 well polystyrene EIA plates (VWR).
• Fifty uL of Ovrl 15 or Pro 104 protein at 0.91 mg/mL in (DPBS) was incubated overnight at 4°C in wells of 96 well polystyrene EIA plates. Plates were washed twice with Tris buffered saline with 0.05% Tween 20, pH 7.4 (TBST).
• the plate wells were then emptied and nonspecific binding capacity was blocked by completely filling the assay wells with TBST/0.5% bovine serum albumin (TBST/BSA) and incubating for 30 minutes at room temperature (RT).
• the plate wells were then emptied, 50 ⁇ L of hybridoma culmre medium samples was added to the wells and incubated for 1 hour at RT.
• the wells were then washed 3 times with (TBST).
• Fc alkaline phosphatase conjugated goat anti-mouse IgG
• hybridomas with supematants producing ELISA absorbance values greater than 1.0 with Ovrl 15 serine protease domain and less than 0.2 with Pro 104 were re-anayed from twenty-five 96 well culmre plates, into new 96 well culmre plates and culmred for a further week.
• MAbs were further screened for reactivity with the human serine proteases testisin, pancreatic trypsin, lung tryptase and kallikrein (Cal Biochem, San Diego, CA) and plasmin and urokinase (American Diagnostica, Greenwich, CT), and mouse testisin, and if reactive with any of these serine protease were excluded from further characterization.
• LMTK-Ovrl 15-HA stable transfectants stable transfectants were grown in DMEM/ 10% FBS + P/S. One day prior to staining, the cells were stimulated by adding sodium butyrate to a 5mM final concentration. LMTK-Ovrl 15-HA cells were washed once with 10ml Ca +2 /Mg +2 free DPBS and then 7ml of warm (37°C) Cellstripper (Mediatech, Hemdon, NA) was added per 150cm 2 flask. The cells were then incubated for 5 minutes at 37°C 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. Human 293F cells were transiently fransfected as described above. The cells were incubated at 37°C for a 30 min. recovery period. Prior to staining, viability of the cells was measured using Guava Niacount (Guava Cytometers, City, CA) and if > 90% viable they were distributed into 96-well v-bottom plates (NWR) for staining with MAbs.
• Guava Niacount Guava Cytometers, City, CA
• Results demonstrating cell surface binding of several of the D-series and F-series MAbs by FACS analysis are listed in Tables 1A, B, C and D below. Results of representative experiments demonstrating cell surface expression by FACS analysis are depicted in Figures 1A and IB. Binding of the MAb Ovrl 15.D3, followed by binding of the donkey anti-mouse Ig-PE conjugate (DAMPE) resulted in 94 % of Ovrl 15 transfected mouse LMTK cells being positive, with a fluorescence intensity (mean fluorescence) 49- fold higher than cells stained with DAMPE alone.
• DAMPE donkey anti-mouse Ig-PE conjugate
• Ovrl 15.D3 and Ovrl 15.D43 bound significantly to Ovrl 15 transfected mouse LMTK cells (Table 1A)
• several more cloned D-series MAbs (Ovrl 15.D15.3, Ovrll5.D32.2, Ovrll5.D37.1, Ovrll5.D43.1 and Ovrll5.D84.2) bound strongly (MFI was from 4 to 76-fold higher than the isotype control) to greater than 50% of Ovrl 15 transfected 293F cells, but not significantly to the non-transfected 293F cells (Table IB).
• Ovrll5.F79 and Ovrll5.F83 bound strongly (MFI was from 4 to 131-fold higher than the isotype control) to greater than 50% of Ovrl 15 transfected 293F cells, but not significantly to the non-transfected 293F cells (Table IC).
• Table 1A demonstrates that monoclonal antibodies Ovrl 15.D3 and Ovrl 15.D20 bound to greater than 50% of the Ovrl 15-transfected LMTK cells. Additionally, Ovrl 15. D3 and Ovrl 15.D20 bound with more than 4-fold higher intensity to Ovrl 15- trnasfected 293F cells than non-transfected 293F cells.
• Ovrl 15.D3 bound with about 1.5-fold higher intensity than the positive control anti-HA MAb.
• Table IB demonstrates that monoclonal antibodies Ovrl 15.D15.3, Ovrll5.D32.2, Ovrl 15.D37.1, Ovrl 15.D43.1 and Ovrl 15.D84.2 all bound to greater than 50% of the Ovrl 15-transfected 293F cells. Additionally, Ovrl 15.D15.3, Ovrl 15.D32.2, Ovrll5.D37.1, Ovrll5.D43.1 and Ovrll5.D84.2 all bound with more than 4-fold higher intensity to Ovrl 15-trnasfected 293F cells than non-transfected 293F cells.
• Table IB results indicate that these MAbs, in particular Ovrl 15.D15.3, Ovrll5.D32.2, Ovrll5.D37.1, Ovrll5.D43.1 and Ovrll5.D84.2, are suitable for immunotherapy of Ovrl 15 expressing cells with or without conjugated drags, toxins, enzymes, prodrag activating molecules or isotopes.
• Table IC demonstrates that monoclonal antibodies Ovrll5.F4, Ovrll5.F9, Ovrll5.F14, Ovrll5.F21, Ovrl l5.F22, Ovrll5.F23, Ovrll5.F24, Ovrll5.F28, Ovrll5.F30, Ovrll5.F31, Ovrll5.F32, Ovrll5.F33, Ovrll5.F35, Ovrll5.F37, Ovrl l5.F43, Ovrll5.F46, Ovrll5.F47, Ovrl l5.F53, Ovrl l5.F54, Ovrll5.F55, Ovrll5.F61, Ovrl l5.F62, Ovrl l5.F63, Ovrll5.F64, Ovrll5.F69, Ovrll5.F71, Ovrll5.F72, Ovrll5.F74, Ovrll5.F75,
• Ovrll5.F14, Ovrll5.F53, Ovrll5.F55, Ovrll5.F64, Ovrll5.F71, Ovrll5.F74 and Ovrl 15.F75 all bound with more than 15-fold higher intensity to Ovrl 15-transfected 293F cells than non-transfected 293F cells.
• Ovrll5.F14, Ovrll5.F53, Ovrll5.F55, Ovrll5.F64, Ovrll5.F71, Ovrll5.F74 and Ovrl 15.F75 all bound with more than 15-fold higher intensity to Ovrl 15-transfected 293F cells than the negative isotype control.
• Table IC results indicate that these MAbs, in particular Ovrl 15.F14, Ovrl 15.F53, Ovrll5.F55, Ovrll5.F64, Ovrll5.F71, Ovrll5.F74 and Ovrl l5.F75 are suitable for immunotherapy of Ovrl 15 expressing cells with or without conjugated drugs, toxins, enzymes, prodrug activating molecules or isotopes.
• Table ID demonstrates that monoclonal antibodies Ovrll5.D20.1, Ovrl l5.F30.1, Ovrl 15.F75.1 and Ovrl 15.F76.2 all bound to greater than 50% of the A431 mmor cells. Additionally, Ovrl 15.D84.2 bound to greater than 30% of the A431 tumor cells.
• Table ID results indicate that these MAbs, in particular Ovrl 15.F30.1, Ovrl 15.F75.1 and Ovrl 15.F76.2, are suitable for immunotherapy of tumors with or without conjugated drags, toxins, enzymes, prodrag activating molecules or isotopes.
• the isotypes of the MAbs were determined using commercially available mouse monoclonal antibody isotyping immunoassay test kits (IsoStrip, Roche Diagnostic Corp., Indianapolis, IN). Results of the isotyping are listed in Table 2.
• Binding kinetics and affinity constants were calculated from surface plasmon resonance measurements using a BIACORE 3000 instrument (Biacore, Piscataway, NJ). Experiments were designed to simultaneously generate on rate, off rate, and affinity values for the Ovrl 15 MAbs.
• Rabbit anti-mouse IgG Fc antibody (Biacore) was immobilized on flow cells 2, 3, and 4 of a CM5 sensor chip (Biacore) by standard amine coupling (Biacore). Flow cell one was used as a blank surface for reference subtractions, and was activated and then inactivated with ethanolamine.
• Ovrl 15 MAbs were captured on the rabbit anti-mouse-IgG Fc coated chip, followed by binding of the antigen.
• MAbs were diluted in HBS EP buffer (Biacore) to 15 ug/mL and were divided into multiple tubes to minimize evaporation between cycles. The MAbs were passed through the flow cells for 2 minutes at 20 uL/minute. The MAb capmre level ranged between 200 and 300 response units (RU) per flow cell. Following MAb capmre the surface was allowed to stabilize for 3 minutes.
• HBS EP buffer Biacore
• Ovrl 15B (1.56mg/mL) antigen was then flowed over the captured MAbs at 20 uL/minute in flow cells and through the blank flow cell, for 4 minutes, at successive concentrations of 144, 72, 36, 18, 9, 4.5 ug/mL. Since the Ovrl 15 molecular weight is 35 kD these antigen concentrations conespond to 4.11, 2.06, 1.03, 0.514, 0.257, 0.129 uM. Two replicates cycles were performed for each antigen concentration or buffer.
• a dissociation time of 420 seconds was allowed between cycles and regeneration of the chip surfaces to anti-mouse IgG Fc antibody or blank surface, were performed by flowing 100 mM Glycine pH 1.75 through the flow cells for 30 seconds at 100 uL/minute.
• the resulting data were analyzed by BiaEvaluation software (Biacore) using a global fit simultaneous ka/kd assuming Langmuir binding.
• the Rmax parameter of the software was set to local to allow compensation for minor variations in the anti-mouse IgG Fc capmre step.
• the calculated affinities presented in Table 3, which are in the 10 " to 10 " M range, are sufficiently high to achieve a therapeutic dose in-vivo at less than or equal to 10 mg/kg.
• Protein extracts for western blot analysis were prepared in cell lysis buffer (1% NP-40, lOmM Sodium Phosphate pH 7.2, 150mM Sodium Chloride) from Ovrl 15-293T transient transfectants and mammalian adenocarcinoma cell lines. Proteins were separated by electrophoresis on NuPAGE 4-12% Bis-Tris gels (Invitrogen Life Technologies, Carlsbad, CA) under denaturing conditions in Novex-XCell II Minicell gel apparatus (Invitrogen, Life Tech) and subsequently transfened to PVDF membranes using an XCell II Blot Module (Invitrogen Life Technologies).
• the membranes were blocked in 1% blocking reagent (Cat#l 096 176, Roche Diagnostic Corp., Indianapolis, IN) and incubated overnight at 4°C with purified primary antibodies (Ovrl 15 monoclonal antibodies: A2.1 , Al 1.1 , A51.2, A52.1 and A63.2 ) and then with horseradish-peroxidase conjugated goat anti-mouse IgG (Cat.#l 15-036-062, Jackson Immunoresearch Laboratories, Inc.) and finally visualized by chemiluminescence using an ECL advance western blotting detection kit (Cat. #RPN2135, Amersham Biosciences, Piscataway, NJ) .
• ECL advance western blotting detection kit Cat. #RPN2135, Amersham Biosciences, Piscataway, NJ
• Ovrl 15 Mab A52.1 did not detect any specific protein bands and was therefore eliminated from further smdies. Since the full length form of Ovrl 15 observed in the SDS-PAGE was larger than the predicted molecular weight of ⁇ 48kDa, we deglycosylated whole cell lysates of Ovrl 15 transfected 293T cells with PNGase F. This treatment shifted the migration of full length Ovrl 15 protein from ⁇ 50kDa to ⁇ 48kDa as detected by Ovrl 15 Mabs A51.2 and A63.2. Deglycosylation did not affect the molecular weight of the 28kDa protein. This is consistent with the observation that no predicted N-glycosylation sites are present in the serine-protease catalytic subunit of Ovrl 15.
• Ovrl 15 was analyzed in pancreatic adenocarcinoma cell lines MiaPaca2, Panel, PL45 as well as in colon adenocarcinoma cell line HCTl 16.
• a band of ⁇ 28 kDa was observed in MiaPaca2 which was absent in the rest of the cell lines analyzed. This directly conelates with data obtained from immunofluorescence studies (Example 2) wherein the highest degree of internalization of Mab A51.2 was observed in MiaPaca2 cell lines with little or none observed in the rest of the cell lines analyzed in this study.
• OvCar-3 ovarian OvCar-3, ovarian CaOV-3, TOV-112D, TOV-21G, SKON-3; pancreatic MIA Paca-2, AsPC-1, PA ⁇ C-1, PL-45, CaPan2; colon HCTl 16, HT29 and breast SKBr-3.
• Ovarian OvCar-3, ovarian CaOV-3, TOV-112D, TOV-21G, SKON-3 cells, pancreatic CaPan2 cells, and colon cells HT29 express Ovrl 15. Control colon HCTl 16 cells do not express Ovrl 15.
• Ovrll5 MAbs of the A-series (Ovrll5.A2.1; Ovrll5.All.l; Ovrll5.A51.2; Ovrll5.A52.1; Ovrl l5.A63.2), D-series and F-series (Ovrll5.F9.2; Ovrll5.F21.1; Ovrll5.F30.1; Ovrll5.F32.2; Ovrl l5.F55.2; Ovrl l5.F62.2; Ovrl l5.F64.2; Ovrll5.F74.2; Ovrll5.F75.1; Ovrll5.F76.2; Ovrll5.F79.2) were tested to demonstrate binding of the antibodies to the cell surface of Ovrl 15 expressing cancer cells by fluorescence microscopy.
• the cells were mounted in Vectastain, a medium containing DAPI (Vector, Burlingame, CA) to visualize the cell nuclei and provide a counterstain, and observed in a Zeiss Fluorescence Microscope Axiophot equipped with the appropriate fluorescent filters. Micrographs were obtained with a CCD camera. Of the five A-series MAbs tested (Ovrl 15 A2.1 ; Ovr 115 Al 1.1 ; Ovrl 15 A51.2. ;
• Figure 2 shows the binding of Ovrl 15 A51.2 to the surface of ovarian CaOV-3 cancer cells. Most cells in the field showed specific labeling for Ovrl 15 could clearly be seen decorating the cell membrane of the cells (anows).
• the D-series MAbs were tested for binding to viable, non-permeabilized Ovrl 15 fransfected 293T cells by fluorescence microscopy.
• the D-series MAbs were further tested by fluorescence microscopy for binding to viable, non-permeabilized Ovrl 15 transfected 293T cells and to mmor cell lines that are either QPCR positive (+) or negative (-) for Ovrl 15 mRNA.
• Table 3B shows that Ovrl 15.D15.3, Ovrll5.D37.1, Ovrll5.D69.1 and Ovrl 15. D81.3 all bound to a significant proportion of the transfected cells and did not significantly bind to the non-transfected 293T cells.
• Ovrll5.D20.1 bound to 20-30% of the QPCR positive mmor cell line SKBR3. Additionally, Ovrl l5.D15.3, Ovrll5.D20.1, Ovrll5.D31.1, Ovrll5.D71.1 and Ovrll5.D81.3 all bound to between 10 and 50% of cells of the QPCR positive tumor cell line HT29.
• Table 3B indicate some differences in MAb specificity for recombinantly expressed and mmor expressed Ovrl 15, which was also in agreement with the FACS results in Example 1. TABLE 3B: BINDING OF Ovrl 15 D-SERIES MAbs TO TRANSFECTED 293T CELLS
• the F-series MAbs were tested by fluorescence microscopy for binding to mmor cell lines that are either QPCR positive (+) or negative (-) for Ovrl 15 mRNA.
• Table 3C shows Ovrll5.F64.2, Ovrll5.F74.2, Ovrll5.F75.1 and Ovrll5.F76.2 all bound to between 20 and 100% of the two QPCR positive mmor cells (HT29 and CaPan2), but not to the QPCR negative cell line HCTl 16.
• the immunofluorescence data in Table 3C complements the FACS data in Example 1. Specifically that Ovrll5.F75.1 and Ovrl 15.76.2 demonsfrated strongly positive results for binding to the QPCR positive mmor cell line A431.
• Ovrl 15 D-series and F-series MAbs demonstrated cell surface binding to significant numbers of tumor cells expressing Ovrl 15 mRNA by fluorescence microscopy. The immunofluorescence intensity was sufficient to clearly observe the cell surface binding. This immunofluorescence data demonstrates these MAbs are suitable for immunotherapy of Ovrl 15 expressing cells with or without conjugated drugs, toxins, enzymes, prodrug activating molecules or isotopes.
• Cy3 Conjugation Ovrll5 A2.1; Ovrll5 All.l; Ovrll5 A51.2; Ovrl l5 A63.2 MAbs were labeled with Cy3. Cy3 conjugation was carried out according to standard procedures and the manufacturer's guidelines. Briefly, 1 mg of antibody was dialyzed against 0.1M bicarbonate buffer (pH 9.3) for 60 min, mixed with Cy3 dye and incubated at RT for 2 hr, and then transferred in a Pierce Slide-A Lyzer Dialysis cassette for dialysis in 2 liters of PBS for 6 hr at 4°C. The operation was repeated 6 times. The Cy3 conjugated antibodies were recovered and concentration was measured in a spectrometer at 280nm.
• Ovrl 15 A2.1; Ovrl 15 Al l.l; Ovrll5 A51.2; Ovrl 15 A63.2 MAbs were then incubated at a concentration of lOug/ml with the cells at 37°C in a water chamber for 60 min, washed in PBS and fix with 3% formaldehyde in PBS for 10 min. Following fixation, the coverslips with the cells were mounted in a medium containing DAPI (Vectastain) to visualize cell nuclei, and observed in a Zeiss fluorescence Microscope Axiophot equipped with the appropriate fluorescent filters. Micrographs were obtained with a color CCD camera.
• DAPI Vectastain
• FIG. 3 A shows the binding of Cy3-A51.2 to the cell surface of ovarian CaOV3 cells, a cell line that expresses Ovrl 15 (anows). Numerous internalization vesicles were detected in the cytoplasm of the cells. Cy3-A51.2 did not bind to the non- expressor control cells HCTl 16 (Fig. 3B). Following binding Cy-Ovrl 15 A51.2 was internalized by ovarian cancer cells.
• FIG 4 shows Cy3-Ovrl 15A51.2 internalization in ovarian TOV-112D (Fig 4A) and TOV-21G (Fig 4B) cancer cells. Numerous internalization vesicles could be seen in the cytoplasm. In addition, a perinuclear labeling conesponding to the presence of endosomes in the vicinity of the Golgi apparams could be seen (anows).
• Figure 5 shows the binding (Fig. 5 A and Fig. 5C) and internalization of Cy3-A51.2 (Fig 5B, anows) in three out of four pancreatic cell lines.
• the binding pattern conesponded to discrete cell surface domains (Fig. 5A and Fig. 5C, anows).
• the internalization pattern staining was characterized by the presence of perinuclear vesicles likely to conespond to endosomes located in the proximity of the Golgi apparams (Fig 5B, anows). Cy3-A51.2 did not bind the pancreatic cell line PL-45, probably because this cell line does not express Ovrl 15 (Fig 5D).
• Ovrl 15 MAbs are internalized in vitro upon binding to Ovrl 15 on the cell surface of Ovrl 15 expressing cancer cells.
• Oyrl 15 distribution in mmors and normal tissues assessed by Immunohistochemistry Tissues: Formalin fixed paraffin embedded blocks of breast, ovarian cancer and normal adjacent tissues were obtained from National Disease Research Interchange (Philadelphia, PA). OCT embedded blocks of normal organs were obtained from Zoion (Hawthorne, NY). Immunohistochemical staining for formalin fixed paraffin embedded sections
• Slides were cut in the cryochamber at 5-8um at an appropriate temperature, air dried for a minimum of thirty minutes at room temperature. Briefly, slides were rinsed in TBS to remove off OCT and incubated at room temperature. IHC was performed using the Immunovision Powervision Kit (Immunovision Technologies,Co. Daly City, CA). Briefly, slides were rinsed in TBS-T to remove off OCT and incubated with a series of different primary antibodies to Ovrl 15 for 1 hour at room temperature. They were then post-fixed in 4% paraformaldehyde fixative for 10 minutes at room temperature and treated as described above.
• the sections were treated with a secondary Cy3 -labeled donkey anti-mouse (Jackson Immunoresearch Laboratories, West Grove, PA) at a concentration of lOug/ml for 30min.
• a secondary Cy3 -labeled donkey anti-mouse Jackson Immunoresearch Laboratories, West Grove, PA
• the clinical sample slides were mounted in Vectastain, a medium containing DAPI (Vector, Burlingame, CA) to visualize the cell nuclei and observed in a Zeiss Fluorescence Microscope Axiophot equipped with the appropriate fluorescent filters. Micrographs were obtained with a CCD camera.
• FIG. 6 and 7 show the distribution of Ovrl 15 in ovarian mmors as evaluated by immunofluorescence using Ovrl 15. A51.2, Ovrl 15.D 15 and Ovrl 15.D84.
• the position of the nuclei of the ovarian cancer cells that are stained in Figures 6A and 6B are shown in Figures 6C and 6D, respectively.
• a strong immunofluorescent signal could be seen on the cell surface of the epithelial cells, indicating that Ovrl 15 is expressed on the cell surface.
• Figure 8 A shows the IHC results obtained with Ovrl 15.D84 in ovarian cancer clinical samples. Three out of five ovarian clinical samples (60%) showed strong labeling on the cell surface of the epithelial cells of the mmor (anows). No specific labeling was observed when normal mouse IgG was used instead of Ovrl 15.D84 (Fig. 8B).
• FIG. 9A shows the immunolabeling pattern obtained using Ovrll5.D84 in clinical samples of pancreas adenocarcinoma. The labeling was restricted to the cell surface of epithelial cells (anows). No specific labeling was observed when normal mouse IgG was used instead of Ovrl 15.D84 (Fig. 9B).
• Figures 10A and 10B demonstrates the labeling of a frozen section from a serous ovarian papillary adenocarcinoma, by Ovrl 15.D43.1 and the lack of labeling in frozen sections of normal human heart, liver and kidney.
• Ovrl 15.D84 The cell membrane of both ovarian and pancreatic epithelial cells was clearly labeled by Ovrl 15.D84, indicating that, following its synthesis, Ovrl 15 is transported to the cell surface. Ovrl 15 expression was also analyzed in normal tissues from a number of vital organs and found to not exceed background.
• Table 4A shows that Ovrll5.F21.1, Ovrll5.F30.1, Ovrl l5.F64.2, Ovrll5.F74.2, Ovrl 15.F75.1, Ovrl 15.F76.2 and Ovrl 15.F 79.2 all reacted with frozen sections from ovarian cancer tissues, but not with frozen sections from normal heart, liver, kidney or ovary.
• Ovrll5.F30.1, Ovrl l5.F32.2, Ovrll5.F74.2 and Ovrll5.F75.1 reacted in more than 50% of the ovarian cancer specimens.
• Ovrl 15.F64.2, Ovrll5.F74.2, Ovrll5.F75.1, Ovrll5.F76.2 and Ovrl l5.F 79.2 were tested on a more extensive panel of frozen normal tissues (Table 4B, below), at 10 ⁇ g/ml, Ovrll5.F64.2, Ovrll5.F74.2, Ovrll5.F75.1, Ovrll5.F79.2 again did not exceed background labeling in frozen sections of tissues from normal heart, liver, kidney, stomach, bladder, testis, colon, ovary, prostate, pancreas, lung or breast.
• Ovrl 15.F76.2 similarly, had no reactivity in these normal tissues, excepting for a weak labeling of the cytoplasm, but not the cell surface membranes, with a low percentage of cells, in one normal pancreas. Many of these tissues are known to contain related serine proteases, such as corin, hepsin and testisin, which further indicated the high specificity of the Ovrl 15 F-series MAbs for the Ovrl 15 protein.
• Ovrl l5.F64.2, Ovrll5.F74.2, Ovrll5.F75.1, Ovrll5.F76.2 and Ovrl 15.F 79.2 were then tested in sections from formalin fixed, paraffin embedded (FFPE) tissues and the results are summarized in Table 4C below.
• Ovrl 15.F74.2 and Ovrl 15.F79.2 both reacted with FFPE sections from 2/4 patients with ovarian cancer, but not with FFPE sections from normal organs, excepting for some weak reactivity of Ovrll5.F79.2 in cardiac muscle, which may be an artifact of fixation, since labeling was not evident in frozen sections of cardiac muscle.
• Ovrll5.F74.2 and Ovrl 15.F76.2 reacted with FFPE sections from 1/4 patients with ovarian cancer, but not with FFPE sections from normal organs.
• Ovrl 15 is expressed in a high percentage of ovarian and pancreatic cancer cases. The fact that Ovrl 15 is expressed on the cell surface of cancer cells makes it an ideal target for antibody based therapy. Several Ovrl 15 MAbs presented above are specific for Ovrl 15 and do not react with fresh frozen tissues from normal vital organs.
• staining is characterized by cell type, staining score (+, ++, or +++), and positives/samples.
• the following abbreviations are used to indicate cell type; Epi: epithelium, Ca: cancer cell.
• table 4D indicates that normal adjacent and normal colon tissue staining is of similar intensity to colon cancer tissue, the pattern of normal adjacent and normal colon tissue staining is more apical and focal or restricted compared to the ubiquitous staining in colon cancer tissues.
• Figures 11 A and 1 IC demonstrate positive membrane staining specifically in colon cancer cells with Ovrll5.F64.2 in frozen colon adenocarcinoma and Ovrll5.D43.1 in FFPE colon adenocarcinoma, respectively. Furthermore, Figures 1 IB and 1 ID display no positive staining in colon cancer when Mouse IgGl replaced Ovrl 15.F64.2 in frozen colon adenocarcinoma and Mouse IgGl replaced Ovrl 15.D43.1 in FFPE colon adenocarcinoma tissue. These results indicate Anti-Ovrl 15 antibodies specifically bind to colon cancer cells expressing Ovrl 15.
• Ovrl 15 MAbs provide adequate specificity to target mmors for immunotherapy with or without attached drags, toxins, enzymes, prodrug activating molecules or isotopes.
• High binding polystyrene plates (Corning Life Sciences (MA)) were coated overnight at 4°C with 1 ⁇ g/well of anti-Ovrl 15 MAb.
• the coating solution was aspirated off and free binding sites were blocked with 300 ⁇ l/well Superblock-TBS (Pierce Biotechnology, Illinois) for lhour at room temperature (RT). After washing 4 times with TBS/0. l%Tween20, lOO ⁇ l of Assay Buffer (TBS buffer pH 7.4, 1% BSA, 1% Calf
• Fifty ⁇ l of unlabeled "coating" MAb (20 ⁇ g/ml) was added and incubated with shaking at RT. After 10 minutes, 50 ⁇ l of biotinylated "detecting" MAb (2 ⁇ g/ml) was added to each well and incubated for 1 hour at RT with shaking.
• the results of the checkerboard ELISA testing 5 MAbs from the A-series, 2 MAbs from the D-series and 13 MAbs from the F-series are shown in the table above.
• the antibodies were tested as coating as well as detecting antibody in all possible combinations. The results are shown as specific signal/ noise ratio. Underlined values in Table 5 above indicate a favorable signal to noise ratio for that MAb pair.
• a 10-fold higher concentration of coating antibody was added to the well to prevent self-pairing. Self-pairing can be observed when antigens are partly multimerized and can confound epitope mapping results. Testing by competitive/blocking ELISA assay assures that antibodies cannot bind to the same epitope when the antigen is in multimeric form.
• the MAbs Ovrll5.D71 and Ovrl 15.F21 may react with epitopes which are proximal to that of Ovrll5.A63.2, since these MAbs in combination result in a lower signal, whereas the other epitopes are apparently all unique from each other and from the Ovrl 15.D71/Ovrl 15.F21 and Ovrl 15 63.2 epitopes.
• the epitope map of the Ovrl 15 MAbs derived from the results in Table 5 is shown in Figure 12. In summary, we can observe that the Ovrl 15 MAbs detected at least four distinct epitopes from this pairing analysis.
• High binding polystyrene plates (Coming Life Sciences (MA)) were coated overnight at 4°C with 1 ⁇ g/well of anti-Ovrl 15 MAb. Free binding sites were blocked with 300 ⁇ l/well Superblock-TBS (Pierce Biotechnology, Illinois) for lhour at room temperature (RT). After washing 4-times with TBS/0. l%Tween20, lOOul of Assay buffer and 20ul of standard (250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 and 0 ng/ml Ovrl 15) or sample were added for 1 hour incubation. After washing, lOOul of biotinylated antibody (lug/ml) was added for 1 hour incubation.
• FIG. 13 demonstrates the detection of the Ovrl 15 protein by sandwich ELISA using various MAb pairs in medium (supernatant) and cell extracts from Ovrl 15 transfected 293T cells and in extracts from the Ovrl 15 mRNA positive mmor cell lines CaOv3, LNCAP and LoVo.
• Substrate Screening Fluorescent protein substrates (TR-X or FL labeled BSA, casein and ovary albumin) and rhodamine B labeled peptides were purchased from Molecular Probes (Eugene, OR) to screen Ovrl 15 specificity. Assay buffers were used following the manufacturer's protocols and varied depending on the substrate. Generally, assays were carried out in 10-20 mM Tris/HCl pH 7.5. The final concentration for protein substrates was 10 ⁇ g/ml and 100 ⁇ M for peptide substrates. The final concentration for Ovrl 15 was 34 ⁇ g/ml (0.73 ⁇ M). All assays were carried out in 96 well-plates.
• Reactions were monitored by following fluorescence reading (Ex 590 nm/Em 650 nm for TR-X label and Ex 485 nm/Em 530 nm for FL and rhodamine B labels).
• Substrate screening indicated rhodamine 110, bis-(CBZ-L-phenylalanyl-L-arginine amide), dihydrochloride; also known as (CBZ-Phe-Arg) 2 -Rl 10 or FR, to be readily hydrolyzed by Ovrl 15.
• Ovrll5 activity The figure demonstrates that the MAbs Ovrl l5.F9.2, Ovrll5.F21.1, Ovrll5.F30.1, Ovrl l5.F32.2, Ovrll5.F55.2, Ovrl l5.F62.2 and Ovrl 15.F76.2 at a concentration of 0.19 ⁇ M (0.5 times mol. excess) significantly reduce Ovrl 15 activity compared to Ovrl 10 antibodies (positive control). Higher concentrations over anti-Ovrl 15 antibodies resulted in further reduction of Ovrl 15 activity. This reduction of Ovrl 15 activity may be due to blocking of the protease active site, steric hindering of Ovrl 15, or conformational change of the Ovrl 15 protein.
• Ovrll5.F9.2, Ovrll5.F21.1, Ovrll5.F30.1, Ovrll5.F32.2, Ovrll5.F55.2, Ovrll5.F62.2 and Ovrll5.F76.2 significantly reduces the activity of Ovrl 15.
• the anti-Ovrl 15 activity screening data demonstrates Ovrl 15 MAbs are suitable for immunotherapy of Ovrl 15 expressing cells with or without conjugated drags, toxins, enzymes, prodrug activating molecules or isotopes.
• Example 5 Functional Validation of Ovrll5
• RK3E, HT-29, CAPAN-2 and HCT-116 cell lines were purchased from American Type Culmre Collection (Manassas, VA). Cells were grown in DMEM (Invitrogen) with L-glutamine plus 4.5g/L glucose and supplemented with 10% FBS and lOOU/mL Penicillin/ Streptomycin (Cellgro). All cells were maintained in a humidified 37°C incubator with 5% C0 2 .
• Extracts from mmor cell or virus-infected cell monolayers were prepared on ice using solubilization buffer (1% NP40, lOmM Na 2 P0 4 , 0.15M NaCl ) plus a protease inhibitor cocktail (Roche Inc.). Protein extracts from human tumors or harvested mmors from xenograft experiments were prepared by homogenization of snap-frozen, minced tumor tissue in extraction buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM EDTA, 0.5% IG-Pal plus protease inhibitors) followed by sonication and then centrifugation in a microfuge to clarify the extracts.
• solubilization buffer 1% NP40, lOmM Na 2 P0 4 , 0.15M NaCl
• Protein extracts from human tumors or harvested mmors from xenograft experiments were prepared by homogenization of snap-frozen, minced tumor tissue in extraction buffer (50
• Membranes were incubated with primary antibody overnight in 5% nonfat dry milk in PBS with 0.05% Tween-20.
• a mouse monoclonal antibody directed against Ovrl 15 (F21.1) was produced in house and was used at a final concentration of lug/ml.
• membranes were washed four times 10 min. at room temperamre in IX PBS with 0.05% Tween-20.
• Horseradish peroxidase linked goat anti-mouse immunoglobulin (Jackson Lab Inc.) was used (1:10,000 dilution) in 5% nonfat dry milk in PBS plus 0.05% Tween-20 for 1 hour at room temperamre to detect the primary monoclonal antibody.
• Membranes were washed four times 10 min. in IX PBS including 0.05% Tween-20 followed by detection using enhanced chemiluminescence (ECL) reagent per manufacturer's directions (Amersham) and exposure to X-ray film (Kodak).
• ECL enhanced chemil
• Figure 15 demonstrates the Ovrll5MAbs, specifically Ovrll5.F21.1, recognizes native Ovrl 15 protein in human cell lines and ovarian mmors.
• a cDNA encoding Ovrl 15 was obtained by PCR from a mmor cDNA preparation. The cDNA was cloned into the Hpa I cloning site of the pLXSN vector (BD Bioscience/Clontech) and sequence verified. A mutant Ovrl 15 protein lacking protease activity was constructed by using oligonucleotide directed mutagenesis to convert the catalytic triad serine residue 385 to an alanine. The resulting mutant cDNA was sequence verified and cloned into the pLXSN vector.
• pLAPSN a retroviral expression vector encoding alkaline phosphatase (AP)
• AP alkaline phosphatase
• pLXSN-AP alkaline phosphatase
• a retroviral expression vector encoding activated v-Ras which was used as a positive control for the apoptosis assay, was constructed by subcloning the RasV12 cDNA from pCMV-RasV12 into pLXSN (BD Bioscience/Clontech) .
• Ecotropic viras was used to infect RK3E cells.
• 293T cells were seeded at a density of 8X10 5 cells per well of a 6 well dish onto Biocoat collagen coated plates (BD). Twenty-four hours later cells were transfected with plasmids using Lipofectamine with the addition of PLUS reagent (Invitrogen) according to the manufacturers recommendations.
• Viras plasmid DNA pLXSN-Ovrll5 wild type, pLXSN-Ovrl 15 mutant, pLXSN-AP or pLXSN-Ras plus pVpack-Eco and pVpackGP (Stratagene) were transfected for three hours after which the cells were grown overnight in DMEM containing 20% FBS. The medium was then changed to DMEM supplemented with 10%FBS + 100 U/mL Pen/Strep and virus-containing media were harvested 24 hours later and filtered through a 0.45 ⁇ m polysulfonic filter.
• Polybrene (Hexadimethrine Bromide; Sigma) was added to fresh virus-containing medium at a final concentration of 4 ⁇ g/ml.
• RK3E cells plated the day before at a density of 3X10 5 cells per 100mm 2 dish, were washed once with phosphate-buffered saline including Ca2+ and Mg2+ (cellgro). The viras containing medium was applied directly to the cells and then incubated for 3 hours at 37°C with occasional swirling.
• the medium was replaced by fresh growth medium and the cells incubated at 37 C for 60-72 hours at which point a final concentration of 350ug/mL of G418 sulfate (Cellgro) was added to the growth medium to select for virus-infected cells. Following G418 selection, pools of cells were used for all subsequent experiments. Expression of ectopic proteins in the viras- infected, selected cells was verified by immunoblot and expression of AP was monitored by staining.
• G418 sulfate Cellgro
• Soft Agar Assays were conducted using 6-well plates (Corning).
• the 2 ml bottom agar base layer consisted of 0.8 % agar, 10% FBS in Iscove's medium (Invitrogen). Trypsinized cells were suspended in 0.4 % agar, 10% FBS in Iscove's medium and applied in a 5 ml final volume on top of the solidified base layer.
• a final 2 ml layer consisting of 0.8 % agar, 10% FBS in Iscove's medium was applied on top of the solidified cell layer.
• Figure 16A is a simple schematic of the step taken to perform the soft agar assay.
• Figures 16B and 16C demonstrate significant colony growth of Ovrl 15 expressing cells and limited growth of AP expressing cells. Furthermore, cells expressing mutated Ovrl 15 lacking protease activity only produced limited colonies indicating that Ovrl 15 protease activity is essential for cellular proliferation.
• Figure 16D is a western blot that confirms Ovrl 15 wild type and the Ovrl 15 mutant lacking protease activity are produced by the cells in the soft agar colonies.
• Retroviras-infected, G418-selected pools of RK3E cells expressing either AP or Ovrl 15 were injected subcutaneously into SCID/Beige mice (Charles River Laboratories). Nine mice were used per group and 5 x 10 6 cells in lOO ⁇ l PBS were implanted without using matrigel. Tumor formation was monitored by palpation and caliper measurement and mmor volume was calculated using the formula: (length x width 2 )/2.
• FIG. 17 demonstrates a significant increase in mean mmor volume over time for cells expressing Ovrl 15 compared to cells expressing AP. This data indicates that Ovrl 15 expression and Ovrl 15 activity is results in mmor progression.
• Figure 18 is a western blot that confirms Ovrl 15 was expressed by the mmors developed in the xenograft experiment.
• Apoptosis Assay 2 x 10 5 RK3E cells expressing AP (negative control), Ras (positive control) or
• Ovrl 15 by retroviral infection and selection were suspended in culmre medium without serum and incubated on poly (2-hydroxyethyl-methacrylate) (Sigma) coated plates. After 24 hours at 37°C cells were harvested by centrifugation and evaluated for apoptosis with a "Guava Nexin V-PE Kit” (Guava Technologies Inc.). After washing approximately 105 cells were resuspended in 40 ⁇ l provided buffer and 5 ⁇ l each Annexin V (+) and 7-AAD (- ) were added. Following 20 minutes incubation on ice, cells were analyzed using the Guava PCA machine according to manufacturer's instructions.
• Figure 19 demonstrates that fewer cells expressing Ovrl 15 undergo apoptosis than cells expressing AP (negative control). Furthermore, the low percentage of early apoptotic cells expressing Ovrl 15 is roughly the same as the cells expressing Ras (positive control).
• Example 6 Killing of HT29 Colon Tumor Cells by Incubation with Ovrll5-MAbs and Anti-mouse MAb Saporin Conjugate
• the anti-transferrin receptor MAb 5E9 (ATCC, Manassas, VA) was used as a positive control MAb for killing. Plates were shaken gently for five minutes to mix the reagents and then incubated at 37°C, in 5% C02. On day 5 (72 h), 10 ⁇ L of a of Alamar Blue stock solution (Biosource International, Camarillo, CA) was added to wells of the first set of plates and they were incubated at 37 °C, in 5% C02 for 2-7h.
• Alamar Blue stock solution Biosource International, Camarillo, CA
• results of testing Ovrll5.D20.1, Ovrll5.D84.2, Ovrll5.F21.1, Ovrll5.F30.1, and Ovrl 15.F76.1 are presented in Table 6 above.
• the MAb Zap alone did not inhibit growth of the HT29 cells (0-2% inhibition).
• the inelevant isotype control MAb did not inhibit growth of the HT29 cells.
• Ovrl 15.D20.1, Ovrl 15.D84.2, Ovrl 15.F21.1 and Ovrl 15.F76.1 MAbs produced between 8 to 18% growth inhibition of HT29 mmor cells.
• Ovrl 15.F76.2 in particular, at concentrations of 0.04 and 2.0 ⁇ g/mL together with MAb Zap resulted in 15-18% greater growth inhibition, than MAb Zap alone.
• Ovrl 15.D20.1 at concentrations of 0.08 and 2.0 ⁇ g/mL together with MAb Zap resulted in 10-12% growth inhibition compared to 0% inhibition by MAb Zap alone.
• Ovrl 15 expressing HT29 mmor cells were obtained at concentrations of MAb which are easily achievable in-vivo, for therapeutic purpose.
• concentrations of MAb which are easily achievable in-vivo, for therapeutic purpose.
• Hybridoma cell lines were deposited with the American Type Culmre Collection (ATCC) located at 10801 University Boulevard, Manassas, Virginia 20110-2209, U.S.A., and accorded accession numbers.
• ATCC American Type Culmre Collection
• the following hybridoma cell lines were deposited with ATCC, Ovrl 15 A51.2, Ovrl l5.D20.1, Ovrll5.D84.2, Ovrll5.F21.1, Ovrll5.F30.1 and Ovrll5.F76.2.
• the names of the deposited hybridoma cell lines above may be shortened for convenience of reference. E.g. A01.1 conesponds to Ovrl 15.A01.1. These hybridomas conespond to the clones (with their full names) deposited with the ATCC.
• Table 7 lists the hybridoma clone deposited with the ATCC, the accorded ATCC accession number, and the date of deposit. Table 7: ATCC de osits

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