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Characterizations of a tumor-associated antigen COX-1 Sheu, Fong-Shyong 1991

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Characterizations of A Tumor-Associated Antigen COX by Fong-Shyong Sheu M.D., Chung-Shan Medical College, Taiwan, R.O.C. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Obstetrics and Gynecology) (Reproductive and Developmental Science Program) We accept t h i s thesis as conforming to the required Standard THE UNIVERSITY OF BRITISH COLUMBIA August 1991 (c) Fong-Shyong Sheu, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia/ I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of £>U 4 fe^V^ The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract II By using modified hybridoma technology, monoclonal antibodies against an ovarian tumor c e l l l i n e , 0C-3-VGH, were generated i n Dr. Lee's laboratory. Among these antibodies, RP 215 was shown to react s p e c i f i c a l l y with a tumor-associated antigen, COX-1. On SDS g e l , COX-1 has a molecular weight of 60 KD and exi s t s as an aggregate i n the natural- state. A highly p u r i f i e d COX-1 was obtained mainly by immunoaffirtity chromatography, with RP 215 as the a f f i n i t y ligand, from the shed medium of cultured tumor c e l l s . A s o l i d phase enzyme immunoassay was established using RP 215 as the capturing and the detecting antibody with a s e n s i t i v i t y of 1 AU/ml, This immunoassay k i t could be used to determine the le v e l s of COX-1 i n the culture medium and i n the sera of cancer patients. COX-1 was characterized under a variety of experimental conditions. At temperatures higher than 50°C or i n the presence of t r y p s i n at 37°C, COX-1 immunoactivity was found to decrease with incubation time. However, COX-1 was not affected by incubation with carbohydrate-digestive enzymes including neuraminidase, Beta-galactosidase and fucosidase or carbohydrate modifying agents such as NaI0 4. Concanavalin A had no e f f e c t on the immunoactivity of COX-1 to RP 215. Furthermore, rabbit antisera against COX-1 were raised, and these polyclonal antisera were shown to exhibit s i m i l a r immunoactivity to that of RP 215 monoclonal antibody. Using the established sandwich enzyme immunoassay, serum l e v e l s of COX-1 among patients with ovarian or c e r v i c a l cancers were determined ret r o s p e c t i v e l y through interlaboratory evaluations and collaborations. Compared to those of normal indivi d u a l s and benign tumors, serum le v e l s of COX-1 were s i g n i f i c a n t l y elevated and can be correlated to the progression of the disease among cancer patients. Preliminary data indicated the COX-1 can complement other established tumor markers such as CA 125 for the purpose of monitoring patients with ovarian or c e r v i c a l cancers. IV Contents I. Abstract I I . Table of contents I I I . L i s t of Tables IV. L i s t of figures V. L i s t of Abbreviations VI. Acknowledgement VII. Introduction A. General review B. Cancer markers C. Ovarian cancers D. C l a s s i f i c a t i o n of ovarian cancer markers 1) Oncofetal antigens 2) Carcinoplacental proteins 3) Tumor associated antigens E. Objective of the present work VIII Materials and methods . . . . A. Materials . . . . . . A) Chemicals . . . . . B) Animals and c e l l l i n e s B. Production & characterization of MAb . A) Production of MAb B) Immunohistochemical characterization of MAb . . . . . C. P u r i f i c a t i o n of monoclonal antibody by DEAE column . . . . D. Immunological methods . . . . page II IV VII VIII IX XI 1 1 2 4 10 11 15 17 21 23 23 23 23 24 24 24 25 26 V IX. A) Enzyme linked immunosorbent assay B) Indirect immunofluoscent assay C) Sandwich enzyme immunoassay D) Tissue adsorption experiment E) Dot b l o t assay E. P u r i f i c a t i o n of COX-1 . A) Preparation of immunoaffinity columns B) P u r i f i c a t i o n of COX-1 from shed medium C) SDS PAGE . . . . . D) Western b l o t . . . . F. Characterization of COX-1 antigen A) Thermal s t a b i l i t y of COX-1 . B) E f f e c t of t r y p s i n and other enzymes C) Determination of molecular weight G. Production of polyclonal antibodies Results . . . . . . A. P u r i f i c a t i o n and characterization of monoclonal antibody (RP 215) A) Tissue adsorption experiment B) Immunohistochemical study C) P u r i f i c a t i o n of RP 215 B. P u r i f i c a t i o n of COX-1 C. Characterization of COX-1 A) E f f e c t of heat on COX-1 immunoactivity B) S e n s i t i v i t y of COX-1 to digestive enzymes 50 C) Molecular d i s t r i b u t i o n . . . 50 26 27 28 29 30 30 30 31 31 32 33 33 33 34 35 36 36 36 41 41 44 44 44 D. Assay of COX-1 s o l i d phase sandwich enzyme immunoassay . . . . . E. Characterization of rabbit antisera against COX-1 . . . . . Discussion . . . . . . References . . . . . . I I I . L i s t of Tables Table 1. FIGO staging of ovarian cancer Page 7 VII Table 2. Tumor markers with c l i n i c a l usefulness i n gynecologic cancers . . . . . 12 Table 3. Immunofluorescent assay of tiss u e sections from a l i s t of benign and malignant tumors 42 Table 4. Cross r e a c t i v i t i e s of RP 215 against tumor c e l l l i n e s by ELISA and immunofluorescent method . . . . . . 43 Table 5. P u r i f i c a t i o n of COX -1 from culture shed medium of 0C-3-VGH c e l l s . . . . 45 Table 6. Preliminary c l i n i c a l evaluation of COX-1 among patients with c e r v i c a l cancer, benign tumor or normal healthy subjects (VGH,Taipei) . . . . . . 66 Table 7. Serum l e v e l s of COX-1 of normal cases and patients with ovarian carcinoma, c e r v i c a l carcinoma, p e l v i c benign tumors and other tissue-originated tumor (from Fu-bei Medical University) . . . . . . 67 Table 8. Comparison of serum COX-1 l e v e l s both i n pre-operative and post-operative cases f o r patients with ovarian carcinoma (from Fu-bei Medical University) . . . . IV. L i s t of figures Figure 1-1. Tissue adsorption experiment to demonstrate the t i s s u e - s p e c i f i c i t y of COX-1 . . . . . Figure 1-2. Indirect Immunofluorescent staining of 0C-3-VGH ovarian cancer c e l l s by RP 215 monoclonal antibody Figure 2. SDS gel electrophoresis of COX-1 recovered from RP 215-Immunoaffinity chromatography . . . . Figure 3. Time-dependent immunoactivity of COX-1 at d i f f e r e n t incubation temperatures . . . . Figure 4. Time-dependent e f f e c t of tr y p s i n digestion on COX-1 immunoactivity Figure 5. Sephacryl S-300 gel f i l t r a t i o n chromatography to reveal the molecular size d i s t r i b u t i o n s of COX-1 i n d i f f e r e n t antigen preparation . . . . Figure 6. A t y p i c a l standard curve of COX-1 (in AU/ml) by the solid-phase sandwich enzyme immunoassay . . . . Figure 7. Determination of t i t e r of rabbit anti-COX-1 by ELISA method V. L i s t of Abbreviations AFP Alphafetoprotein ATCC American Type Culture C o l l e c t i o n Inc. AU Arbi t r a r y Unit BSA ' Bovine Serum Albumin CEA Carcinoembryonic Antigen Con A Concanavalin A CPM Count per minute DEAE Dethylaminoethyl EDTA Ethylenediaminetretraacetic Acid ELISA Enzyme-linked Immunosorbent assay EIA Enzyme Immunoassay HCG Human Chorionic Gonadotropin HPL Human Placental Lactogen HRP Horse Raddish Peroxidase IEF Isoelectronic Focusing IgG+M Immunoglobulin G & M KD Kilodalton LB Luria-Bertani MAb Monoclonal antibody O.D. Optical Density PAGE Polyacrylamide Gel Electrophoresis PBS Phosphate Buffered Solution PEG Polyethylene Glycol PI I s o e l e c t r i c Point RIA Radioimmunoassay X RPMI Russell Park Memorial I n s t i t u t e SDS Sodium Dodecyl Sulfate TBS T r i s Buffered Solution TBST TBS + 0.05% Tween-20 T r i s Trihydroxymethylaminomethane u l M i c r o l i t e r XI Acknowledgements I wish to extend my thanks to a l l members regarding my thesis work. F i r s t of a l l , I appreciate my supervisor, Dr. Gregory Lee, who gave me t h i s opportunity to learn basic research technique i n our labs. Secondly, I would l i k e to thank Dr. Basal Ho Yuen (Grace Hospital) and Dr. Diane M i l l e r (VGH) f o r helping me c o l l e c t blood samples for my c l i n i c a l evaluation. Also, I appreciate a l l of my committee members: Dr. Rajamahendran (Chairman of committee, Dept. of Animal Science), Dr. J . T. Emerman (Dept. of Anatomy, UBC), Dr. Y. S. Moon (Dept. of Obstetrics & Gynecology), and Dr. Dun Rurak (Dept. of Obstetrics & Gynecology and Research Center Shaughnessy Hospital) who organized t h i s committee and gave me valuable suggestions. Furthermore, I would l i k e to thank Dr. Angus Tsang fo r h i s advice i n thesis writing and organization, Lebby Balakshin f o r s e c r e t a r i a l assistance, Jennifer Yao for correction, and a l l my co-workers i n our lab during the two years. F i n a l l y , I would l i k e to thank my family - my father who supported my research and unfortunately passed away l a s t year; my wife f o r moral and s p i r i t u a l support and my son, Kevin, f o r his computer work. 1 Introduction A. General review: Cancers are tissues composed of c e l l s that have become independent to some degree of the normal regulation of growth of the host c e l l . Cancer c e l l s are characterized by the tendency to change t h e i r morphologies, biochemical properties and the properties of chromosomal structures. These abnormal c e l l s can p r o l i f e r a t e and produce more and more abnormal c e l l s which subsequently spread to d i f f e r e n t parts of the body, eventually leading to death i f untreated. The incidence of cancer has increased progressively during the l a s t decades, and i t has been claimed that i f the same trend continues, by the end of t h i s century there w i l l be more than f i f t y thousand cancer deaths each year i n the United States alone. To date, our understanding of the regulation of c e l l d i f f e r e n t i a t i o n and development i s s t i l l very li m i t e d and the control mechanism fo r t h i s complex process has not been elucidated. Although much work has been done i n an attempt to i d e n t i f y the regulatory mechanism of the development of cancers, so f a r there have been very few advances. I t i s obvious that i f cancer patients can be diagnosed at ah e a r l i e r stage and prompt medical treatment i s provided, the s u r v i v a l rate of cancer patients could be greatly improved. Over the l a s t twenty years, development i n the area of b i o l o g i c a l research including the discovery of techniques to produce monoclonal antibodies (Kohler and 2 M i l s t e i n , 1975; Minna et a l , 1981.) have opened up a new avenue f o r cancer research. With the aid of t h i s technology, antibodies with a predefined antigen s p e c i f i c i t y can thus be obtained. The hybrid clone that produces the antibody can be amplified to provide a renewable source of the highly monospecific reagent which can be exploited f o r use i n d i f f e r e n t applications i n cancer disease. These applications include diagnosis, imaging and treatment of cancers (Mclntire 1985). B. Cancer markers: The association of b i o l o g i c a l markers with cancers has been recognized for many decades (Jones, 1848; Zondek, 1930; and Gutman et a l , 1936). In c e r t a i n types of cancers including those of ovarian o r i g i n s , tumor c e l l s possess the a b i l i t y to synthesize s p e c i f i c protein molecules that are expressed at the c e l l surface. These molicules are sometimes shed into the serum, and can be detected by means of s p e c i f i c and s e n s i t i v e immunoassay. The f i r s t cancer marker "Bence-Jones protein" was i d e n t i f i e d over four decades ago (Bence-Jones, 1847; Edelman & Poulik, 1961; Porter, 1963) from patients who suffered from a tumor of plasma c e l l - m u l t i p l e myeloma. Since then, a number of tumor markers including monoclonal immunoglobulins, hormones, secreted serum proteins, s p e c i f i c enzymes and isoenzymes, c e l l surface components, glycoproteins and carbohydrates, etc., have been i d e n t i f i e d by various laboratories. The 3 applications of most of these components are presently being investigated. The c l i n i c a l usefulness of some of them such as CEA, AFP and HCG has already been well recognized ( S e l l , 1990) and are already commercially available ( S e l l , 1990). It has been noticed that the expression of a given cancer marker i s re l a t e d to the tumor's tissu e o r i g i n ( S e l l , 1990). For instance, tumors a r i s i n g from c l o s e l y r e l a t e d embryological tissues, such as g a s t r o i n t e s t i n a l cancers, may a l l produce alpha-fetoprotein (AFP), a substance normally produced by f e t a l l i v e r and yolk sac. On the other hand, evidence obtained indicates that the concentration of a tumor marker i n a patient's body f l u i d might r e f l e c t a dynamic balance that represents a combination of tumor a c t i v i t y and marker turn-over (Accola et a l , 1980). In t h i s aspect, these markers are considered to be a useful aid f o r the assessment of tumor burden. In addition, i t i s well recognized that c e r t a i n tumor markers can provide an accurate and constant estimate of the effectiveness of treatment, and serve as a monitor during active tumor therapy. Most recently, many attempts have been focused on the u t i l i z a t i o n of tumor markers i n targeting antibody-bound cytotoxic agents to p r e f e r e n t i a l l y control or destroy malignant c e l l s ( S e l l , 1990; Nadji, 1990). For cancer therapy, a number of toxins have been used for chemotherapy of cancers and proven to be e f f e c t i v e i n k i l l i n g cancer c e l l s . However, the drawback of using these toxins i s that 4 they also exert a to x i c e f f e c t on p r o l i f e r a t i n g normal c e l l s . Therefore, the potential of using monoclonal antibodies to s p e c i f i c a l l y d e l i v e r anti-tumor drugs, toxins, or radionuclides to cancer has received extensive investigation ( S e l l , 1990; Nadji, 1990) and shed l i g h t on the treatment of cancers. Monoclonal antibodies directed against well-defined tumor associated antigens have made i t possible to evaluate t h e i r s u i t a b i l i t y as c a r r i e r s f o r d i r e c t d e l i v e r y of t o x i c agents to tumor targets. They could also a s s i s t i n the development of a treatment which reduces t o x i c e f f e c t s on normal c e l l s and f a c i l i t a t e s damage on more s e l e c t i v e targets. C. Ovarian cancers: Ovarian cancer i s the fourth most frequent cause of cancer death i n women. Over the past 30 years, the mortality of the patients who suffe r from ovarian cancer has increased considerablely (Green et a l 1984). Although ovarian cancer occurs less frequently than some other kinds of gynecological carcinoma, such as c e r v i c a l or endometrial cancers, they are responsible f o r more deaths (Hart 1981; Young, et a l , 1985). C l i n i c a l data indicates that the f i v e year s u r v i v a l rate of ovarian cancer i s usually r e l a t i v e l y low (20-47%). The high l e t h a l rate of ovarian cancer i s mainly due to the the fac t that the tumor i s often severely advanced by the time of c l i n i c a l diagnosis, and the tumor c e l l s are l i k e l y to have spread throughout the peritoneal cavit y . Further decreases i n the chance of s u r v i v a l are 5 thought to be attributed to the f a c t that most ovarian cancers respond poorly to chemotherapy and radiotherapy (Morrow, 1981; Richardson, 1985b). I t has been reported that among the common female gen i t a l cancers, s i x t y percent of vulvar cancers are diagnosed at stage I & I I . At these stages, the cure rates of these cancers are about 80-90%. For vaginal and c e r v i c a l cancers, more than 50% of the patients can be diagnosed at either stage I or stage II with 5 year s u r v i v a l rates of 85% and 60%, respectively. About 90% of endometrial cancers are detected at stage I & II with a cure rate of 90% for stage I and 57% f o r stage I I . In contrast, less than 50% of ovarian cancers can be diagnosed at stage I or stage I I . In f a c t , approximately 65% of patients with ovarian cancers are already inoperable when f i r s t diagnosed (McGown 1978). I t has been considered that the f a i l u r e of diagnosis of ovarian cancer at a curable stage i s probably because ( i ) the ovaries are r e l a t i v e l y inaccessible f o r accurate evaluation ( i i ) ovarian carcinoma, l i k e other v i s c e r a l malignancies, do not display early symptoms and ( i i i ) screening procedures, such as s p e c i f i c immunologic or biochemical markers i n the blood or urine, have yet to be developed (Lingeman, 1983). On the other hand, i t i s also believed that ovarian cancer usually spreads throughout the peritoneal cavity and forms multiple metastatic nodules which are often undetected by noninvasive techniques. As a r e s u l t , i n the majority of 6 cases, i n i t i a l surgery shows that the tumors have spread beyond the ovaries (Morrow, 1981). There i s a wide range i n the malignant potential of ovarian tumors. The complicated histogensis of ovarian tumors can be understood on the basis of the in d i v i d u a l elements making up t h i s organ. The e p i t h e l i a l c e l l s , growing germ c e l l s and supporting mesenchymal c e l l s of the ovary a l l possess the poten t i a l to become malignant c e l l s . A u n i f i e d c l a s s i f i c a t i o n of cancers has been adopted by the World Health Organization (Scully, 1975) and the International Federation of Gynecology and Obstetrics (FIGO, Table I ) . According to t h i s c l a s s i f i c a t i o n , neoplasms thought to be derived from the ovarian surface epithelium are c l a s s i f i e d as "common e p i t h e l i a l tumours" of the ovary which includes serous, mucinous, endometriods, c l e a r c e l l s and Brenners tumors, s o l i d adenocarcinoma, carcinomas and mixed mullerian tumors (Fenoglio, 1980; Scully, 1977; and Serov, 1973). These tumors are notorious f o r t h e i r wide v a r i a t i o n i n the degree of h i s t o l o g i c d i f f e r e n t i a t i o n that can be found i n d i f f e r e n t areas of the same neoplasm (Hart, 1981). The other two major types of ovarian cancers are those c e l l tumors developed i n the germ c e l l s ( l i p o i d c e l l s tumors) and those developed i n the granulosa and stroma c e l l s (sex cord - stromal tumors). In humans, the most common ovarian cancers (over 80%) are found to be derived from e p i t h e l i a l c e l l s (Servo, 1973; Young et a l , 1985). 7 Table 1 FIGO Staging of Ovarian Cancer* Stage Description Stage I Growth l i m i t e d to the ovaries Stage l a Growth lim i t e d to one ovary; no ascites 1. No tumor on the external surface; capsule i n t a c t 2. Tumor present on the external surface and/or capsule ruptured Stage l b Growth l i m i t e d to both ovaries; no ascites 1. No tumor on the external surface; capsule i n t a c t 2. Tumor present on the external surface and/or capsule ruptured Stage Ic Tumor eithe r stage l a or stage l b , but with ascites present or p o s i t i v e peritoneal washings Stage II Growth involving one or both ovaries with p e l v i c extension Stage I l a Extension and/or metastases to the uterus and/or tubes Stage l i b Extension to other p e l v i c tissues Stage l i e Tumor either stage I l a or stage l i b but with ascites present or p o s i t i v e peritoneal washings Stage III Growth involving one or both ovaries with intraperitoneal metastases outside the p e l v i s and/or p o s i t i v e retroperitoneal nodes; tumor limi t e d to the true p e l v i s with h i s t o l o g i c a l l y proved malignant extension to small bowel or omentum Stage IV Growth involving one or both ovaries with distant metastases; If pleural effusion present, p o s i t i v e cytology required to assign a case to stage IV, parenchymal l i v e r metastases equals stage IV Special category Unexplored cases thought to be ovarian carcinoma *Obtained from "Cancer Diagnosis i n v i t r o using monoclonal antibodies" edited by Herbert Kupchik (pl43) 8 Ovarian cancers can be found at a l l ages from infancy to old age. However, nonepithelial neoplasms account f o r most of the neoplasms i n younger patients, while adenocarcinomas predomimnate i n women of 25 years or older i n most developed countries (Lingeman, 1983). The incidence of e p i t h e l i a l tumor r i s e s r a p i d l y a f t e r age 40 u n t i l a plateau occurs at age 80(Hart, 1981). In general, i t appears that ovarian cancer increases with "ovulatory age" -the t o t a l time i n a woman's l i f e during which her ovarian cycle i s not supressed by pregnancy, l a c t a t i o n or the use of or a l contraceptives (Casagrande et a l , 1979). Also i t has been observed that frequent ovulation could increase the frequency of cyst formation which i s thought to be associated with many incidences of ovarian cancers (Hart 1981). Despite the prevalence of ovarian cancers, knowledge concerning the pathogenesis and etiology of ovarian cancer remains unresolved. General epidemiologic studies suggest that disordered endocrine function may contribute to the development of ovarian cancer (Joly & L i l i e n f i e l d , 1974; Berval et a l , 1978; Lingeman, 1974). Although the rel a t i o n s h i p between ovarian cancer and the administration of synthetic estrogen has not been c l e a r l y defined, i t has been suggested that there may be an increased r i s k i n patients receiving s t i l b e s t r o l f o r menopausal symptoms (Hoover et a l , 1977). On the other hand, considerable 9 evidence c o l l e c t e d implies that some environmental agents such as p o l y c y l i c aromatic hydrocarbons, i n d u s t r i a l pollutants, smoking (MattisonS Thorgeirrsson, 1978), asbestos (Newhous, et a l , 1972), t a l c (Cramer, et a l 1982) and a n t i r u s t o i l (Jarvholm, 1981) may be carcinogenic. F a m i l i a l and genetic factors have also been reported to be causes of ovarian cancers. In addition, geographical factors have been taken into account since migrants from low incidence countries to high incidence countries usually develop a s i m i l a r incidence rate as the new host country within one or two generations (Liggeman, 1983). Epidemiologic studies also suggest that cancer of the breast and ovary might share some common e t i o l o g i c f a c t o rs. For instance, women with ovarian cancer could have a 3 to 4 f o l d increase i n the frequency of subsequent breast cancer, and women with breast cancer have a much higher (2 times) chance of developing ovarian cancer (Young et a l , 1985). 10 D. C l a s s i f i c a t i o n of Ovarian Cancer Markers I t i s evident that ovarian tumor c e l l s possess the a b i l i t y to synthesize s p e c i f i c protein molecules that are expressed at the c e l l surface and sometimes shed into the serum ( S e l l , 1990; Nadji, 1990). If a sen s i t i v e assay of a tumor marker could be developed f o r the detection of ovarian cancers at an e a r l i e r stage, the 5-year su r v i v a l rate of patients could be su b s t a n t i a l l y improved. During the past years many e f f o r t s have been made to i d e n t i f y a r e l i a b l e serum marker f o r ovarian cancers. Although some of them have been commercially available, none i s t o t a l l y s a t i s f a c t o r y . Recently, with the application of advanced hybridoma technology, monoclonal antibodies (MAbs) with high s p e c i f i c i t y can be established to react with a d i s t i n c t antigenic determinant or epitope present on p a r t i c u l a r antigen molecules, thus providing a more s p e c i f i c and sens i t i v e t o o l f o r cancer detection (Accola et a l , 1980; Knauf & Urbach, 1980; Bast et a l , 1981; Bhattacharya et a l , 1982; Kabawat et a l , 1983; Tagliabue at a l , 1985; T s u j i et a l , 1985; M i o t t i et a l , 1985; Malnguene, 1986; Kawahara et a l , 1986; Fleuron et a l , 1987; Thor et a l , 1987). 11 Potential tumor markers for ovarian cancer so f a r i d e n t i f i e d can generally be c l a s s i f i e d into three broad categories based on t h e i r nature (table 2). These are (1) oncofetal antigens; (2) carcinoplacental proteins; and (3) tumor associated antigens. (1) Oncofetal antigens: The application of cancer markers as a more generally applicable t o o l began with the discovery of AFP and carcinoembryonic antigen (CEA). (Gold & Freedman, 1965; Hoskins et a l , 1987). Carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) are examples of oncofetal antigens which are produced only by normal embryonic tissues during f e t a l l i f e . When a mature c e l l undergoes neoplastic transformation, the production of these proteins become reactivated as a r e s u l t of the loss of regulator gene function. CEA i s a large family of related c e l l surface glycoproteins that have a domain structure s i m i l a r to that of immunoglobulins (Thimpson, 1988). CEA was f i r s t i d e n t i f i e d by Gold and Freedman (1965) as an antigen s p e c i f i c f o r adenocarcinomas of the digestive t r a c t . I t s chemical structure i s very complicated, and the CEA family consists of about 10 genes. So f a r , up to 36 d i f f e r e n t glycoproteins have already been i d e n t i f i e d . Chemically, CEA i s a complex glycoprotein with numerous antigenic s i t e s and a molecular weight ranging from 180,000 - 200,000 daltons . The r a t i o of protein to carbohydrate varies from 1:1 to 1:5 i n 12 Table 2 Tumor markers with c l i n i c a l usefulness i n gynecologic cancers* I. Oncofetal antigens A. Carcinoembryonic antigen (CEA) B. a-Fetoprotein (AFP) II . Carcinoplacental antigens A. Human chorionic gonadotropin (HCG) B. Human placental lactogen (HPL) C. Regan isoenzyme (RI) I I I . Tumor-associated antigens A. Ovarian cystadenocarcinoma antigen (OCAA) B. Ovarian cancer antigen (OCA) C. NB/70K D. CA 19-9 E. CA-125 *Obtained from "Tumor markers and tumor associated antigens" by Bismal C. Ghosh, Luna Gosh (1987) 13 CEA from d i f f e r e n t tumors (Go & Ammon, 1975) The protein portion of the singl e polypeptide chain appears to account for the major antigenic determinants (Pritchard & Todd, 1979). CEA i s synthesized by columnar e p i t h e l i a l c e l l s and concentrated i n the glycocalyx along the luminal border or extravascular body f l u i d s (Martin F. et a l , 1973, Fle i s h e r M. et a l , 1973, Wahren B. et a l , 1975). In ovarian carcinomas, i t has been reported that more than 46% of patients have elevated CEA l e v e l s i n t h e i r plasma (Van Nagell et a l , 1981). Measurable l e v e l s of CEA have been reported i n 50% of patients with stage III e p i t h e l i a l ovarian cancer. I t has been found that the frequency of elevated CEA l e v e l s progressively increases with advancing stage and bulk of tumor. Although CEA i s detectable i n a l l the major c e l l types of e p i t h e l i a l ovarian cancers, i t can also be observed i n mucinous ovarian tumors (Heald et a l , 1978; Charpin et a l , 1982). Besides oVarian cancers, CEA i s also found i n a large v a r i e t y of cancers of e p i t h e l i a l o r i g i n (Mclntire, 1985). Furthermore, the finding of elevated CEA i n nonmalignant disease conditions and even i n smokers (Hansen et a l , 1974) has la r g e l y been a problem i n applying t h i s marker to screen large populations i n an attempt to detect s u b c l i n i c a l ovarian cancer (Van Nagell et a l , 1976; Hansen et a l , 1974; Smith & O i l , 1984). The elevated l e v e l of CEA can therefore only be considered as a suggestive parameter 14 i n cancer diagnosis (Go, 1976; Zamcheck, 1981; Staab, 1984 and Terry et a l , 1974). Currently, the major usefulness of CEA i s l i m i t e d to the follow-up of ovarian cancer patients whose plasma l e v e l s are elevated p r i o r to therapy. Alpha-fetoprotein (AFP) i s another oncofetal protein f i r s t described by Abelev et a l (Abelev, 1963). I t i s o r i g i n a l l y produced during f e t a l development by the l i v e r , yolk sac, and g a s t r o i n t e s t i n a l epithelium. AFP i s also a secretory protein synthesized by tumor c e l l s (Tatarinov, 1964 and Mclntire, 1975). This property makes AFP an ideal tumor marker since i t can be detected i n the c i r c u l a t i o n of patients. I t i s a single chain glycoprotein with a molecular weight of 69,000 daltons. The carbohydrate content of AFP i s about 4% of the molecular weight (Ruoslahti, et a l , 1979). AFP exhibits microheterogeneity from d i f f e r e n t sources because of v a r i a t i o n i n s i a l i c acid content (Ruoslahti et a l , 1979). Generally, a value of 20 to 25 ng/ml or greater of AFP i n serum i s considered elevated (Donaldson et a l , 1980). Similar to that of CEA, elevations of serum AFP i n ovarian cancer patients have also been reported (Esterhay et a l , 1973; Talerman et a l , 1974; Ghosh P.157 41, 42). Serum AFP i s elevated i n most cases of endodermal sinus tumor of the ovary i n which a sample i s taken p r i o r to s u r g i c a l excision of the cancer (Kurman et a l , 1976; G a l l i o n et a l , 1979). I t i s reported that serum AFP l e v e l s r e f l e c t 15 c l i n i c a l disease status i n more than 75% of cases of endodermal sinus tumor (Wilkinson et a l , 1973; Homey et a l , 1975). Therefore, the s e r i a l measurement of AFP i s considered to be very h e l p f u l i n postsurgical evaluation of therapy for patients with endodermal sinus tumors of the ovary (Goldstein & Piro, 1972). Patients with ovarian teratocarcinomasa also show elevated serum AFP, but i t i s unclear i f these teratomas contain endodermal sinus tumor elements (Esterhay et a l , 1973, Talerman et a l , 1974). However, i n 53 patients with e p i t h e l i a l carcinoma of the ovary, a l l had AFP l e v e l s lower than the cut o f f range (25 ng/ml), except one patient with a tumor of uncertain o r i g i n (Seppala et a l , 1975). In addition, AFP elevation i n the serum of the patients with benign conditions, such as acute v i r a l h e p a t i t i s , l i v e r c i r r h o s i s , obstructive jaundice, and hepatic trauma, i s also observed. In t h i s regard, AFP i s not an e n t i r e l y r e l i a b l e indicator of ovarian malignancy (Abelev, 1971). Nonetheless, from the c l i n i c a l data obtained, AFP i s s t i l l accepted as being useful i n diagnosing endodermal sinus tumors i n women with a ra p i d l y enlarging s o l i d ovarian mass. (2) Carcinoplacental proteins: Carcinoplacental proteins are protein moieties, e.g. human chorionic gonadotropin (HCG), normally produced by placental tissues during pregnancy. These proteins are also found i n c e r t a i n r a p i d l y growing neoplastic c e l l s (Van 16 Nagell et a l , 1981; 1982). HCG i s a well known hormonal glycoprotein composed of two subunits, the alpha and beta chains, and a molecular weight of approximately 39,000 daltons. HCG or i t s beta-subunit i s a valuable marker fo r ovarian germ c e l l tumors that contain either embryonal c e l l carcinomas or choriocarcinoma (Kurman et a l , 1976). Although HCG can, to a c e r t a i n extent, r e f l e c t the status of ovarian germ c e l l tumors, i t i s considered to be of l i t t l e value i n patients with ovarian cancers of e p i t h e l i a l o r i g i n (van Nagell et a l , 1981). Human placental lactogen (HPL) i s found normally i n the human placenta and i s detected i n a high percentage of patients with gestational trophoblastic disease. Although HPL i s observed occasionally i n the serum of patients with e p i t h e l i a l and germ c e l l ovarian cancers, i t s usefulness i n patients with gyneocologic malignancy i s very l i m i t e d (Stuhlmill & Seigler, 1987). Regan isoenzyme (RI) i s an a l k a l i n e phosphatase found i n normal placenta (Stolbach et a l , 1969; Rosen, 1975) with a molecular weight of 120,000 daltons. I t i s detected i n a number of gynecologic cancers, and 47% of patients with ovarian carcinoma show elevated l e v e l s of RI (Kellen et a l , 1976). However, the effectiveness of RI i n cancer diagnosis i s l i m i t e d as elevated serum l e v e l s of RI are also found i n patients with diseases such as h e p a t i t i s , c o l i t i s , f a m i l i a l 17 polyposis, and even i n smokers (van Nagell et a l , 1976; Smith & Oi, 1984). (3) Tumor Associated antigens: I t i s known that i n many types of cancer tumor c e l l s possess the a b i l i t y to synthesize s p e c i f i c proteins which can be used to d i s t i n g u i s h the neoplastic c e l l s from normal c e l l s ( S e l l , 1990; Nadji, 1990). Ovarian cancer c e l l s also express or secrete unique glycoproteins that can p o t e n t i a l l y be used f o r immunodiagnosis and immunotherapy (Kenemans et a l , 1985; Kenemans et a l , 1988). I t i s evident that a l l benign and malignant e p i t h e l i a l tumors of the ovary including serous, mucinous, endometrioid, c l e a r c e l l and t r a n s i t i o n a l types express two or more proteins s i m i l a r to keratin. However, by judging the p r o f i l e of t h i s p a r t i c u l a r antigen, ovarian e p i t h e l i a l neoplasm cannot be d i f f e r e n t i a t e d from e p i t h e l i a l neoplasms of other organs (Dabbs & Geisinger, 1988). In addition to k e r a t i n - l i k e antigens, e p i t h e l i a l tumors of the ovary usually contain other e p i t h e l i a l markers, namely, e p i t h e l i a l membrane antigen (EMA) and human milk f a t glo b u l i n (HMFG) membranous antigens. Similar to that of keratin, these two antigens are not unique to ovarian cancer tissues (Nadji, 1990) and can also be found i n other e p i t h e l i a l neoplasms as well as i n malignant mesotheliomas. 18 Ovarian cystadenocarcinoma antigen (OCAA) i s a glycoprotein chemically d i f f e r e n t from CEA and AFP and unique to neoplasmic ovarian tissues (Bhattacharya & Barlow, 1975). Sixty percent of early stage ovarian cancer patients i s reported to have elevated serum OCAA l e v e l s , and up to 80% of patients with advanced disease exhibits high l e v e l s of OCAA (Bhattacharya & Barlow, 1979). Serum concentration of OCAA i n ovarian cancer patients c o r r e l a t s with the tumor volume and the status of the disease. In patients with tumor completely resected, the serum l e v e l returns to normal shortly a f t e r operation. However, t h i s antigen i s not s p e c i f i c enough to be used as a screening t e s t f o r ovarian cancer, since 35% of patients with cancers not of ovarian o r i g i n , such as breast and c o l o n i a l cancers also exhibit a pos i t i v e reaction to antisera against OCAA (Bhattacharya & Chatterjee, 1980). OCA (Ovarian cancer antigen) i s another tumor associated glycoprotein. F i r s t described by Knauf and Urbach (Knauf & Urbach, 1977; 1981), Sixty percent of patients with ovarian cancer are observed to have elevated OCA l e v e l s i n t h e i r plasma. However, more than 10% of patients with benign gynecologic disease or who are pregnant are also detected to be p o s i t i v e (Knauf & Urbach, 1980). Due to the lack of s p e c i f i c i t y of OCA, i t i s excluded as an e f f i c i e n t screening t o o l . 19 NB/70K i s an ovarian tumor-associated antigen i d e n t i f i e d from ovarian cancer patients by radioimmunoassay (Knauf & Urbach, 1981). I t i s reported that NB/70K i s a glycoprotein with a M.W. of 70,000 daltons and i s present i n most e p i t h e l i a l ovarian adenocarcinomas but not i n normal tissues of the ovaries ( B r i z z a r i et a l , 1983). The p o s i t i v e predictive rate f o r ovarian cancers using polyclonal antisera or monoclonal antisera against t h i s antigen i s not promising (Ken et a l , 1981; Knauf & Urbach, 1981). A number of tumor-associated antigens f o r ovarian cancers are also reported by various groups of researchers (Barlow & Bhattacharya, 1983; Cordon-Cardo et a l , 1985; Mariani-Costantini, et a l , 1985), including CA 19-9 (Finkler et a l , 1988), TA-4 (Kato & Torigoe, 1977) TAG-72 (Thor et a l , 1986), OV632 (Kaelma et a l , 1988), MH 94 CI (Nouwen et a l , 1987) and MF 116 (Nouwen et a l , 1987). However, most of these markers are not ovary-specific, and can be detected i n other types of tumors and even i n normal t i s s u e s . Recently, two monoclonal antibodies(MAb) namely OC 125 which reacts with antigen CA 125 and OWTL3 which reacts with antigen OA 3, are considered to be the most promising markers with respect to c l i n i c a l a p p l i c a b i l i t y i n gynecological oncology (Kenemans, 1990). OV-TL 3 MAb was established by fusion of murine myeloma c e l l s with spleen c e l l s of mice immunized with ovarian endometriod cancer c e l l s (Bast et a l , 1981). OV-TL 3 seems to be highly 20 s p e c i f i c f o r ovarian e p i t h e l i a l tumors, and useful f o r the d i f f e r e n t i a l diagnosis, e s p e c i a l l y of c o l o r e c t a l cancer (Henzen-Logmans et a l , 1988). This may be due to the f a c t that OA 3 antigen exhibits a high expression exclusively i n ovarian cancers. However, OA3 antigen i s membrane bound and not shed into the blood serum. Therefore, although i t has a high s p e c i f i c i t y to ovarian cancer, the f e a s i b i l i t y of using 0V-TL3 antibody for serum immunoassay becomes p a r t i c u l a r l y l i m i t e d . CA 125 i s an antigen associated with ovarian carcinoma c e l l s (Davis, et a l , 1986). A MAb OC 125, raised by immunizing mice with an e p i t h e l i a l ovarian cancer c e l l l i n e (OVCA-33) i s o l a t e d from the ascites f l u i d of a cancer patient, can i d e n t i f y t h i s antigen CA 125 i s reported to be a glycoprotein with a molecular weight greater than 500,000 daltons (Bolts & D ' l n c l c i , 1978). The MAb OC 125 i s found to recognize the CA 125 antigenic determinant present on the glycoprotein complex (Bast, et a l , 1981). The nature of the epitope recognized i s not known but i t could be a carbohydrate moiety of the protein molecules. CA 125 i s detected i n f e t a l mullerian epithelium, peritoneum, pleura, pericardium and amnion (Kabawat et a l , 1983). Immuno-histological s t a i n i n g of frozen tumor specimens indicats that 77% of the nonmucinous ovarian tumors including serous, endometriods and c l e a r c e l l types are p o s i t i v e (Castaldo, et a l , 1981). The antigen i s not 21 detected i n normal ovarian tissues and only 1% of apparently healthy person have elevated antigen l e v e l s . Changes i n serum CA 125 l e v e l s are found to correlate with disease progression or regression i n more than 90% of cases studied. Although CA 125 appears to have a high s e n s i t i v i t y for ovarian cancers, elevated l e v e l s are also detected i n some benign diseases and i n other e p i t h e l i a l cancers (Breast and lung cancers) (Endo et a l , 1988). Furthermore, most Krukenberg's tumors express detectable CA 125 (Fukazawa et a l , 1988). About 25% and 5% of patients with nongynecologic malignancies and benign disease, respectively, are reported to have elevated antigen l e v e l s . The e f f i c a c y and r e l i a b i l i t y of CA 125 i n the early diagnosis and monitoring of patients with ovarian carcinoma i s s t i l l under active investigation. E. Objective of the present work: Over the past few decades, much research e f f o r t has been directed toward searching f o r a s p e c i f i c tumor marker for ovarian cancers. Although a number of tumor markers have been i d e n t i f i e d and studied, so f a r , none of them appears to be s a t i s f a c t o r y . In t h i s regard, intensive research i s , therefore, es s e n t i a l for the i d e n t i f i c a t i o n of a new tumor-associated antigen which p o t e n t i a l l y can be used for the development of a diagnostic aid for the detection of ovarian cancer. 22 With the application of hybridoma technology, i t i s possible to es t a b l i s h monoclonal antibodies (MAb) with high s p e c i f i c i t y to react with a d i s t i n c t antigenic epitope present on a p a r t i c u l a r antigen molecule, thus providing a more s p e c i f i c t o o l f o r cancer detection (Accola, et a l , 1980; Kawahara, et a l , 1986). A monoclonal antibody, RP 215, has recently been developed i n our laboratory, which i s highly s p e c i f i c to a tumor-associated antigen (COX-1) i d e n t i f i e d from an ovarian cancer c e l l l i n e (0C-3-VGH) (Chao, et a l . , 1983). In the present work, e f f o r t s are directed toward the i s o l a t i o n , p u r i f i c a t i o n and characterization of the antigen COX-1 from the shed culture medium of ovarian cancer c e l l s , OC-3-VGH. In addition, attempts are made to determine the biochemical nature of the determinant recognized by the MAb RP 215 and to develop an immunoassay f o r t h i s antigen. The usefulness of t h i s assay k i t i s being evaluated by various groups of collaborators and t h e i r preliminary c l i n i c a l data w i l l be presented and discussed i n the th e s i s . 23 VIII. Materials and Methods A.Materials: A) Chemicals: Tissue culture media and supplements including RPMI 1640, IMDM, glutamine and p e n i c i l l i n - streptomicin (100X) were from Gibco, Burlington, Ontario, Canada; 2,6,10,14-tetramethy-pentadecane (pristane), dimethylsulfoxide (DMSO), methylcellulose, bovine serum albumin (BSA), polyethylene g l y c o l (PEG), complete and incomplete Freund's adjuvant were purchased from Sigma Chemical Company, St Louis, MO; fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG+A+M was from Cappel, Malvern, PA; Reagent f o r sodium dodecyl s u l f a t e (SDS) acrylamide gel electrophoresis were from Bio-Rad Laboratories, Richmond, CA; Iodine-125 radioisotope was from Amersham, Oakville, Ontaria, Canada. B) Animal and c e l l l i n e s : Inbred male mice of BALB/C s t r a i n (6-10 weeks) were from Charles River, Canada; the ovarian cancer c e l l s l i n e , 0C-3-VGH, was of serous o r i g i n and was established by the Department of Obstetrics and Gynaecology, Veterans General Hospital, T a i p e i , Taiwan (Chao et a l , 1983). Several other c e l l l i n e s including ME-180 (human epidermoid c e r v i c a l cancer), Shiha (human c e r v i c a l squamous carcinoma), JEG (human choriocarcinoma), C33A (human c e r v i c a l carcinoma) and AN3A (human endometrial carcinoma) 24 were obtained from American Type Culture C o l l e c t i o n Inc. (ATCC, Rockville, Maryland). B. Production and characterization of monoclonal antibody (RP 215) A) Production of monoclonal antibodies Monoclonal antibodies could be produced either from culture medium of antibody-secreting hybrid c e l l s which were the r e s u l t of previous c e l l fusion screened i n Dr. Lee's lab, or from ascites f l u i d c o l l e c t e d from BALB/C mice primed with pristane according to the published procedures (Freund, et a l , 1982). B r i e f l y , pristane (0.5 ml) was injected 5 days i n advance to the intraperitoneal cavity of the BALB/c mice followed by intraperitoneal i n j e c t i o n of 1 x 10 5 to 1 x 10 6 antibody-secreting hybrid c e l l s i n serum free medium. The ascites f l u i d was drawn af t e r one week and stored at -20°C. B) Immunohistochemical characterization of monoclonal antibody, RP-215 Formalin-fixed, praraffin-embedded normal human ti s s u e sections were used to determine the s p e c i f i c i t y or cross-r e a c t i v i t y of the generated monoclonal antibodies to various tissues including brain, l i v e r , heart, kidney, spleen, ovary, t e s t i s , cervix, and muscle. These tissues were obtained at the time of surgery or at autopsy. P a r a f f i n 25 tissue sections were deparaffinized i n xylene, dipped i n 95%, 75% and 50% ethanol, and f i n a l l y washed with PBS. Cryostat tis s u e sections were also prepared and dipped i n 95% ethanol followed by wash with PBS. The immunofluorescent method was performed according to procedures to be described l a t e r (p27). Several c e l l l i n e s , including ME-180, Shiha, Jeg, C33A and AN3A, were examined f o r t h e i r respective cross r e a c t i v i t i e s to the RP 215 monoclonal antibody. For immunofluorescent study, 50 u l of cultured c e l l s with a concentration of l x l 0 6 / m l from each c e l l l i n e were dried on the s l i d e s . The s l i d e s were then blocked with blocking solut i o n containing 0.5% BSA i n PBS and fix e d with methanol. Twenty u l of RP 215 from the culture supernatant was added, followed by one hour incubation. After washing three times with PBS-BSA, 20 u l of goat-anti-mouse Ig+A+M-FITC was added. For ELISA, the c e l l extract from each c e l l l i n e was adjusted with PBS to 0.05 absorbance/ml at 280 nm. Details of the assay procedures w i l l be described l a t e r (p26). C. P u r f i c a t i o n of RP 215 by DEAE column: F i f t y ml of ascites f l u i d containing RP 215 monoclonal antibody was f i r s t p r e c i p i t a t e d with 50% saturated ammonium su l f a t e . The suspension was centrifuged at 10,000 rpm f o r ten minutes. The p e l l e t was redissolved i n 20 ml of d i s t i l l e d water. The protein solution was then p r e c i p i t a t e d 26 again with 40% ammonium su l f a t e and centrifuged at 10,000 rpm f o r 10 minutes. The p e l l e t was redissolved i n about 10 ml of 10 mM T r i s buffer, pH 8.0 and dialyzed overnight against the same buffer at 4°C. The p r e c i p i t a t e d protein was removed by centrifugation. The d i a l i z e d solution was loaded (1 mg protein/ml gel) onto a DEAE column (size 2.5 x 30 cm) which had been equilibrated with 10 mM T r i s buffer pH 8. After loading, the column was washed with the same buffer u n t i l absorbance at 280 nm decreased to blank l e v e l . The solution was then eluted with a l i n e a r gradient of 0 to 0.3 M NaCl i n 10 mM T r i s HC1, pH 8.0. The f r a c t i o n s containing the highest absorbance at 280 nm were c o l l e c t e d . The purity of RP 215 was analyzed using SDS-PAGE with 10% acrylamide gels(see p31). D. Immunological methods: A) Enzyme-linked immunosorbent assay (ELISA) The s p e c i f i c binding of monoclonal antibody RP 215 to OC-3-VGH c e l l s was also determined by ELISA (Lee C.Y.G. et a l , 1984). B r i e f l y , c e l l s from OC-3-VGH c e l l l i n e were cultured i n each of 96 wells i n m i c r o t i t e r plates u n t i l a monolayer was formed. A l t e r n a t i v e l y , OC-3-VGH c e l l s could be harvested from c e l l cultures and dried on m i c r o t i t e r wells at a f i n a l concentration of 1 x 10 6/ml. After removal of culture supernatant, the cell-coated wells were blocked by incubation with PBS-BSA (0.5% BSA i n PBS) f o r one hour p r i o r 27 to use by ELISA. After one hour of incubation with the t e s t antibody i n culture supernatant, wells were washed three times with PBS-BSA. Goat anti-mouse IgG+M+A conjugated with horse radish peroxidase was added to each well f o r an additional hour incubation at room temperature. Following three washes with PBS-BSA, 3,3',5,5'- tetramethyl benzidine (0.3 mg/ml) and 0.02% H 20 2 i n 0.1 M citrate-phosphate buffer, pH 5.0 were added to i n i t i a t e the enzymatic reaction at room temperature f o r 15 minutes. The color reaction was stopped by adding 1 M H 2S0 4. The color i n t e n s i t y i n each well was determined spectrophotometrically at 450 nm by a CLS microplate reader (Cambridge, UK). Normal mouse serum i n RPMI 1640 medium served as the negative control i n a l l assays, while mouse antisera against 0C-3-VGH c e l l s served as the p o s i t i v e c o n t r o l . B) Indirect immunofluorescent assay Indirect immunofluorescent assay was used to determine the binding of antibodies to the COX-1 c e l l surface antigen and to determine antibody t i t e r s . This assay was performed using methanol-fixed tumor c e l l s according to the reported procedure (Bast at e l , 1984). Due to the f a c t that methanol-fixed c e l l s or p a r a f f i n embedded tissue sections do not consistently preserve t h e i r surface antigens which react with monoclonal antibodies, an i n d i r e c t immunofluorescent assay using a l i v e c e l l suspension was also performed. B r i e f l y , 50 u l of c e l l culture supernatant containing 28 monoclonal antibodies was mixed with a suspension of cultured 0C-3-VGH c e l l s (1 x 10 5/ml) i n either microfuge tubes or m i c r o t i t e r wells. The mixture was incubated at room temperature for 3 hours. Following three washes with PBS-BSA and centrifugation, FITC-labeled goat anti-mouse IgG+M+A was added to the c e l l suspension and the mixture was incubated f o r an additional two hours. Following three washes with PBS-BSA and centrifugation, the f i n a l superatant was discarded. F i n a l l y , 10 u l of 80% g l y c e r o l i n PBS was added to the c e l l p e l l e t and transferred to s l i d e s for observation under a fluorescence microscope. C) Sandwich enzyme immunoassay One of the monoclonal antibodies, RP 215, was shown to recognize repeated epitopes of the complementary tumor-associated antigen Cox-1. Therefore, t h i s antibody was used to determine Cox-1 l e v e l s i n the sera of ovarian cancer patients and those of normal healthy subjects by means of sandwich enzyme assay. B r i e f l y , RP 215 was p u r i f i e d from ascites f l u i d by ammonium sulf a t e fractionations and DEAE ion exchange chromatography. P u r i f i e d monoclonal antibody was coated on microwells at a concentration of 5 - 10 ug/ml i n 50 mM Tris-HCl, pH 8.0. At the same time, RP 215,labelled with HRP (horse radish peroxidase), at a concentration of 1/400 with conjugate buffer (1% BSA, 0.2% PEG, 0.1% Thimersol i n 0.15M T r i s pH 8.0) was added to the wells, and t h i s served as the second antibody tracer i n the sandwich 29 enzyme immunoassay. The immunoassay was i n i t i a t e d by incubating 100 u l of human serum and RP 215 labeled HRP (1:400) i n antibody-coated microwells (Duplicate) f o r three hours at room temperature. After incubation, the content of the microwells was removed by suction. The wells, as i n previous procedures, were washed three times with PBS-tween 0.02% and twice with d i s t i l l e d water. PBS containing 5% BSA served as negative cont r o l . Concentrate shed medium from 0C-3-VGH pre c i p i t a t e d with ammonium s u l f a t e served as standard for the assay of Cox-1. D) Tissue adsorption experiment Normal human tissues such as l i v e r , kidney, t e s t i s , muscle, heart and brain were homogenized. The concentration was adjusted to 10 mg/ml with 1% BSA i n PBS. F i f t y u l of tissue homogenate sample was added to mic r o t i t e r wells coated with 0C-3-VGH c e l l s . One hundred u l of RP 215-HRP (horse radish peroxidase) of d i f f e r e n t d i l u t i o n s was combined with 0.2 M T r i s HC1, pH 8, 50% newborn serum or c a l f serum, 0.1 % thimersol and phenol and added to each well. For the control, 50 u l of 1% BSA i n PBS or COX-1 p a r t i a l l y p u r i f i e d from shed medium of 0C-3-VGH c e l l were used as negative and po s i t i v e c o n t r o l . The a c t i v i t y was determined by EIA as described. 30 E) Dot blot assay The dot b l o t assay was modified from the method described by Towbin (Towbin et a l . , 1979). This method would give rapid detection of antigen i n various samples and was e s p e c i a l l y useful f o r t e s t i n g column eluents during antigen p u r i f i c a t i o n . B r i e f l y , 5 u l of sample was dotted onto n i t r o c e l l u l o s e f i l t e r paper and allowed to dry completely. The n i t r o c e l l u l o s e f i l t e r was blocked with 5 % BSA i n PBS f o r 1 hour, then incubated with iodinated monoclonal antibody RP 215 f o r another two and a h a l f . After washing three times with TBST, the n i t r o c e l l u l o s e f i l t e r was counted on a gamma counter. E. P u r i f i c a t i o n of COX-1 A) Preparation of Immunoaffinity column: Monoclonal antibodies were coupled to affini-Gel-10 (BioRad Co.) according to established protocols. Eight ml of RP 215 (1.3mg/ml i n 0.1M NaHC03, pH 8.3) p u r i f i e d by DEAE column was mixed with 5 ml affini-Gel-10 and shook for two hours at room temperature. The column was washed with PBS and 1M T r i s HC1, pH 8.0, (2 volumns each) and then by 2M NaCl containing 6N urea. F i n a l l y , the column was washed with 2 volumes of 50 mM glycine HC1 pH 2.2 p r i o r to e q u i l i b r a t i o n with PBS f o r antigen p u r i f i c a t i o n . 31 B) P u r i f i c a t i o n of COX-1 from the shed culture medium: COX-1 was p u r i f i e d from shed culture medium by ammonium sul f a t e f r a c t i o n a t i o n and then by immunoaffinity chromatography using RP 215 as the a f f i n i t y ligand. B r i e f l y , COX-1 i n shed medium was f i r s t p r e c i p i t a t e d i n the presence of 35% ammonium s u l f a t e . Following centrifugation, the p e l l e t was redissolved i n minimal amount of PBS and dialyzed overnight against the same buffer. Following centrifugation to remove the denatured protein, the c l e a r supernatant was then passed through a 10 ml RP 215-immunoaffinity column. The a f f i n i t y column was then washed extensively with 300 ml of PBS at a flow-rate of 50 ml/hr. This was followed by a continued wash with an additional 300 ml of PBS containing 1M NaCl to eliminate a l l possible n o n s p e c i f i c a l l y bound proteins. F i n a l l y , COX-1 was eluted with 50 mM glycine HC1, pH 2.2., and f r a c t i o n s of 1 ml were c o l l e c t e d and neutralized immediately with 1M T r i s , HC1, pH 8.0. During the course of antigen p u r i f i c a t i o n , COX-1 immunoactivity i n the shed medium and p u r i f i e d preparation was determined by sandwich enzyme immunoassay. The p u r i t y of COX-1 preparation was examined by SDS gel electrophoresis, two-dimensional gel electrophoresis (O'Farrel et a l , 1977) and by Western blot assay (Lee et a l 1982). C) SDS-PAGE (SDS polyacrylamide Gel Electrophoresis) The purity of the f r a c t i o n s containing the highest OD 32 at 280 nm and immunoactivity was studied by 10% SDS-PAGE. Samples mixed with an equal amount of sample buffer (15 ul) were heated f o r f i v e minutes at 100°C to denature the protein and then applied to the g e l . The i n i t i a l voltage for electrophoresis was set at 100 V. When the sample protein reached the top of the running g e l , the voltage was set to 200 V. The electrophoresis was stopped when the dye reached 1 cm from the bottom of the running g e l . After electrophoresis, the gel was removed c a r e f u l l y f o r protein v i s u a l i z a t i o n with eit h e r s i l v e r or coomassie st a i n i n g . D) Western b l o t assay This method was used to i d e n t i f y the s p e c i f i c protein bands seperated by SDS-PAGE i n the presence of antibodies. D e t a i l s of t h i s method are described i n the i n s t r u c t i o n manual supplied by Bio-Rad (Towbin et a l , 1979). The p u r i f i e d antigen was also analyzed by Western b l o t assay. After electrophoresis, the gel and the n i t r o c e l l u l o s e f i l t e r paper were rinsed with transfer buffer. The whole sandwich unit was placed i n the electrophoretic transfer apparatus with the gel facing the cathode. The buffer tank was f i l l e d with enough transfer buffer to cover the g e l . After 2 hours of electrophoretic transfer at a constant voltage of 50V, the n i t r o c e l l u l o s e paper was removed and placed i n blocking buffer f o r one hour. COX-1 which reacted with monoclonal antibodies or Rabbit polyclonal antibodies could be analyzed by either 33 d i r e c t method or i n d i r e c t method. By d i r e c t method, RP 215 conjugated HRP (Horse Radish Peroxidase) i n 1:400 d i l u t i o n was added a f t e r the n i t r o c e l l u l o s e paper had been blocked at 4°C overnight. By i n d i r e c t method, COX-1 reacted with rabbit antisera (1:100) for two hours. After washing with TBST, goat a n t i - r a b b i t IgG conjugated with HRP was added for another two hours. After washing with TBST, the protein pattern was stained by adding enzyme substrates. F. Characterization of COX-1 antigen: A) Thermal S t a b i l i t y of COX-1 Immunoactivity The thermal s t a b i l i t y of COX-1 aggregates was determined by i t s immunoactivity using the established sandwich enzyme immunoassay. B r i e f l y , the tumor c e l l extract or shed medium was incubated at 50 °C or higher temperatures for various time i n t e r v a l s . At the end of each incubation, aliquots were drawn and cooled at 0°C i n i c e . The residual Cox-1 immunoactivity was then determined using the established EIA procedure. B) E f f e c t of t r y p s i n and other hydrolytic enzymes or chemicals on COX-1 Immunoactivity In order to determine whether t r y p s i n could a f f e c t COX-1 immunoactivity, the concentrated shed medium was incubated with t r y p s i n at a concentration of 1 mg/ml for various time i n t e r v a l s at 37°C. At the end of each 34 incubation, t r y p s i n a c t i v i t y was neutralized with two f o l d excess of soybean t r y p s i n i n h i b i t o r (1 mg/ml). The residual COX-1 immunoactivity was determined by the established sandwich enzyme immunoassay. S i m i l a r l y , carbohydrate-digestive enzymes or carbohydrate-binding proteins, namely neuraminidase, beta-galactosidase, fucosidase and Concanavalin A, N-glycosidase and O-glycosidase, were incubated with COX-1 solutions to determine whether COX-1 immunoactivity would be affected. The carbohydrate modifying agent, NAI0 4 ( f i n a l concentration 0.1M) was added to COX-1 solu t i o n ( i n PBS) and incubated for 1 hour at room temperature. At the end of incubation, COX-1 solution was dialyzed extensively against PBS at 4°C. The remaining COX-1 immunoactivity was determined by the established EIA and compared to that of the control i n which no NAI0 4 was added so to determine i t s e f f e c t s . C) Determination of molecular weight of COX-1 i n the native state The molecular weight of COX-1 i n the native state was determined by Sephacryl S-300 gel f i l t r a t i o n chromatography. Myoglobin (Mr. 13,000), ovalbumin (45,000), immunoglobulin G (160,000) and thyroglobulin (675,000) were used as molecular weight standards. By using Sephacryl S-300 gel f i l t r a t i o n chromatography, the molecular weight d i s t r i b u t i o n s of 35 p u r i f i e d COX-1, tumor c e l l extract and concentrated shed medium were determined. B r i e f l y , swollen Sephacryl S-300 i n a 22 ml column (size 1 x 30 cm) was equilib r a t e d with PBS containing 0.1% Thimersol. One ml of p u r i f i e d COX-1, OC-3-VGH c e l l s extract or concentrated shed medium (lOx) was loaded on to the column. Fractions of 0.5 ml were co l l e c t e d . Immunoactivity f o r COX-1 i n each f r a c t i o n was determined by the established sandwich enzyme immunoassay. G. Production of polyclonal antisera against p u r i f i e d COX-1 Antisera against p u r i f i e d COX-1 were raised i n New Zealand white female rabbits according to t y p i c a l immunization protocols (Lee et a l , 1984). B r i e f l y , 50 ug of p u r i f i e d COX-1 i n 0.5 ml of PBS was emulsified with an equal volume of complete Freund's adjuvant. The mixture was injected into the back of the rabbit subcutaneously i n 10 d i f f e r e n t s i t e s . Two weeks a f t e r the primary immunization, the rabbits were immunized twice with the same amount of immunogen except that incomplete Freund's adjuvant was used. One week a f t e r the t h i r d immunization, blood was drawn from the r e t r o a u r i c u l a r vein of the rabbit and the t i t e r was checked by EIA or radioimmunobinding assay using I 125 -labeled COX-1 as the tracer (Lee et a l 1986). 36 1%. Results RP 215 was one of 3000 monoclonal antibodies generated against 0C-3-VGH ovarian cancer c e l l l i n e , i n i t i a l l y , t h i s antibody was selected based on i t s p o s i t i v e binding to OC-3-VGH ovarian cancer c e l l s coated on mic r o t i t e r wells as judged from the r e s u l t s of ELISA. The RP 215-secreting hybrid c e l l l i n e was further established by the a b i l i t y of t h i s antibody to react with l i v e or methanol-fixed OC-3-VGH c e l l s by i n d i r e c t immunofluorescent assay (Figure 1-2). A. P u r i f i c a t i o n and Characterization of monoclonal antibody, RP 215 The t i s s u e - s p e c i f i c i t y of RP 215 was generally determined by tissue adsorption experiments and an immunohistochemical study of human tissue sections to be described as follows: (A) Tissue adsorption experiments (Figure 1-1) To examine the t i s s u e - s p e c i f i c i t y of selected monoclonal antibodies, a quantitative t i s s u e adsorption experiment was performed. The r e s u l t s of t h i s experiment shown i n Figure 1-1 revealed that normal human tissues do not i n t e r f e r e with the binding between HRP - labeled RP 215 and the OC-3-VGH ovarian cancer c e l l s coated on microwells. 37 Figure 1-1 Tissue adsorption experiment to demonstrate the lack of i n h i b i t i o n of horseradish peroxidase-labeled RP 215 (HRP-RP 215) binding to 0C-3-VGH tumor c e l l s coated on microwells by the normal t i s s u e homogenates, where ( • ), ( O ), ( A )' ( .A), ( • ) a n < i ( • ) denote those of kidney, l i v e r , muscle, t e s t i s , heart and brain, respectively. ( ^  ) denotes soluble extract of 0C-3-VGH tumor c e l l s (protein concentration of 10 mg/ml), ( ) denotes the nonspecific binding between HRP-RP 215 and microwells coated with unrelated proteins, and ( X ) denotes the BSA (10 mg/ml). 15h <J0 D I L U T I O N F A C T O R 39 Figure 1-2 Indirect immunofluorescent st a i n i n g of 0C-3-VGH ovarian cancer c e l l s by RP 215 monoclonal antibody observed under fluorescence microscope: (A) under UV l i g h t ; and (B) under v i s i b l e l i g h t . 4-0 o A B 41 (B) Immunohistochemical study: Normal human tissue s l i d e s of vaginal wall, uterine cervix, f a l l o p i a n tube, ovary, heart, colon and some benign and malignant tumors of d i f f e r e n t tissue were deparaffinized and fi x e d i n methanol f o r 5 min and blocked with PBS BSA. Immunohistochemical study was performed by the immunofluorescent procedure. RP 215 was shown to have the highest s p e c i f i c i t y to 0C-3-VGH c e l l s (Figure 1-2) and did not cross react with other normal human tissues or other benign and malignant tumors (Table 3). Several c e l l l i n e s including ME 180, AN3A, Shiha, C33A and Jeg were also used to determine t h e i r respective cross r e a c t i v i t i e s to RP 215 by ELISA and i n d i r e c t immunofluorescent method. I t was observed that RP 215 can recognize antigens only from ME 180 and 0C-3-VGH c e l l s (Figure 1-2) but not any other established c e l l l i n e s used i n t h i s study. The r e s u l t s are presented i n Table 4. (C) P u r i f i c a t i o n of RP 215 monoclonal antibody from a s c i t i s f l u i d From 50 m i n i l i t e r s of ascites f l u i d , about 180 mg of monoclonal antibodies could be p u r i f i e d through the DEAE c e l l u l o s e column. 42 Table 3. Immunofluorescent assay of p a r a f f i n t i s s u e sections from c l i n i c a l l y defined benign and malignant tumors Tissues C l i n i c a l diagnosis Indirect* Immunofluorence muscle f i b r o s i s tongue mucocele -hypopharynx adenoid c y s t i c a Ca -s a l i v a r y gland chronic s i a l o a d e n i t i s -i n t e s t i n e adenocarcinoma -f a l l o p i a n tube ectopic pregnancy + ovary simple cyst -anus hemorrhoid -bursa baker cyst -skin f i b r o s i s -breast f ibroadenoma -spleen spleen rupture — *0C-3-VGH c e l l s f i x e d on s l i d e s were used as the p o s i t i v e control 43 Table 4 Cross r e a c t i v i t i e s of RP 215 with the established tumor c e l l l i n e s by ELISA and immunofluorescent assay c e l l l i n e IIF Assay ELISA with shed medium ELISA With c e l l extract 0C-3-VGH + 1.069 0.439 ME 180 + 0.726 0.349 Shiha - 0.190 0.104 Jeg - O.190 0.128 C33A - 0.173 0.102 AN3CA - 0.170 0.151 Tumor c e l l l i n e s obtained from American type culture c o l l e c t i o n Inc.. 0C-3-VGH - ovarian cancer c e l l s (serous type) Me-180 - human epidermoid c e r v i c a l cancer (ATCC HTB 33) Shiha - human c e r v i c a l squamous carcinoma (ATCC HTB 35) JEG - human choriocarcinoma (ATCC HTB 36) C33A - human c e r v i c a l carcinoma (ATCC HTB 31) AN3CA - human endometrial Ca (ATCC HTB 111) IIF - i n d i r e c t immunofluorescence ELISA - enzyme-linked immunosorbent assay 44 B. P u r i f i c a t i o n of COX-1: The tumor-associated antigen, COX-1 was p u r i f i e d mainly from cultured shed medium by using RP 215 as the ligand i n the immnoaffinity chromatography. The molecular weight of p u r i f i e d COX-1 was shown to be 60 KD on SDS Gels (Fig 2) and confirmed by Western b l o t assay. In the neutral state, p u r i f i e d COX-1 existed as an undefined aggregate of 60 KD basic subunit with molecular weight ranging from 1 x 10 5 to 1.5 x 10 6 daltons. This can be shown by the e l u t i o n p r o f i l e s from Sephacryl S-300 gel f i l t r a t i o n chromatography as shown i n Figure 5. By radioimmuno dot assay the s p e c i f i c a c t i v i t y of p u r i f i e d COX-1 could reach up to 15,000 times that of the s t a r t i n g material (Table 5). The p u r i f i e d COX-1 could be used f o r iodination, standard antigen preparations or f o r immunization i n rabbit to r a i s e polyclonal antibodies. C. Characterization of COX-1 A) Thermal s t a b i l i t y of COX-1 immunoactivity As shown i n Figure 3, COX-1 immunoactivity was r e l a t i v e l y stable i n a PBS solution of neutral pH at temperatures below 50"c. At incubation temperatures higher than 50°C, C0X-1 immunoactivity was observed to decrease s i g n i f i c a n t l y with incubation time. The immunoactivity of COX-1 completely diminished a f t e r incubation at 100°C f o r 10 minutes. 45 Table 5 P u r i f i c a t i o n of COX-1 from culture shed medium of cultured 0C-3-VGH c e l l s P u r i f i c a t i o n protein con centration (ug/ml) s p e c i f i c a c t i v i t y ** f o l d Of protein p u r i f i c a t i o n shed medium 2780 2.3 1 ammonium su l f a t e ppt. 6712 26.3 11 Imraunoaffinity 24)* 11. 25 8853 3849 Chromatography 25)* 10 34720 15056 26)* 15 12186 5298 *Fraction number of immunoaffinity chromatography p r o f i l e **Determined by dot b l o t immunoassay described i n the text 46 Figure 2 Results of SDS gel electrophoresis and the corresponding Western b l o t assay to reveal respectively the purity and immunospecificity of COX-1 p u r i f i e d by immunoaffinity chromatography. Lane B i s SDS gel of p u r i f i e d COX-1 ( 5 ug/well, f r a c t i o n #25 of Table 5); lane A i s COX-1 revealed by Western blot assay; lane C i s the molecular weight standards of the corresponding SDS gel with si z e of molecular weight markers indicated by arrows on the r i g h t . 4-7 A B C MWIK) 48 Figure 3. E f f e c t of temperature and incubation time on the immunoactivity of COX-1. Samples of COX-1 were incubated at d i f f e r e n t temperatures for d i f f e r e n t time i n t e r v a l s , where (#)/ (•), (A) and ( X ) denote 50°C, 60°C, 80°C and 100°C, respectively. The residual a c t i v i t y of COX-1 was determined by the solid-phase sandwich enzyme immnoassay described i n the text. 43 50 B) S e n s i t i v i t y of COX-1 immunoactivity to digestive enzymes, Con A and NaI0 4 and t r y p s i n : Under the assay conditions described, COX-1 immunoactivity was shown to be s e n s i t i v e to p r o t e o l y t i c digestion by t r y p s i n at 37°C (Figure 4) when analyzed by sandwich EIA method. I t was observed that only 0.7% of COX-1 immunoactivity could be recovered a f t e r incubation with t r y p s i n for 60 minutes. However, COX-1 immunoactivity was not affected s i g n i f i c a n t l y by incubation with concanavalin A (con A), NaI0 4 and the carbohydrate-digestive enzymes including neuraminidase, beta-galactosidase and fucosidase, N-glycosidase and O-glycosidase. C) Molecular weight d i s t r i b u t i o n Using Sephacryl S-300 gel f i l t r a t i o n chromatography, the molecular weight d i s t r i b u t i o n of p u r i f i e d COX-1 p u r i f i e d , tumor c e l l extract and concentrated shed medium was determined. The r e s u l t s of t h i s analysis are presented i n Figure 5. 51 Figure 4. E f f e c t of tr y p s i n digestion on COX-1 Immunoactivity. COX-1 antigen was incubated with t r y p s i n at 37° C for the various time i n t e r v a l s as described i n the procedures. The residual a c t i v i t y of COX-1 was determined by the solid-phase sandwich enzyme immunoassay, described i n the text. 53 Figure 5 Sephacryl S-300 gel f i l t r a t i o n chromatography to reveal the molecular s i z e d i s t r i b u t i o n s of COX-1 i n d i f f e r e n t antigen preparations. Relative immunoactivities of COX-1 were determined by the established solid-phase sandwich immunoassay described i n the text. Where ( A ), ( • )/ and ( O ) represent those of COX-1 antigens prepared from crude 0C-3-VGH extract, p u r i f i e d from shed medium, and concentrated shed medium derived from cultured 0C-3-VGH c e l l s , respectively. A gel f i l t r a t i o n column (size 1x30cm) equilibra t e d with PBS-BSA containing 0.1% thimersol was used for t h i s analysis. Fractions of 0.5 ml/tube were c o l l e c t e d and assayed for immunoactivity. The void volume and column volume of t h i s column appeared i n fractio n s #19 and #44, respectively, as judged from the c a l i b r a t i o n with the known molecular weight standards and blue dextran (molecular weight 2x l 0 6 dalton) described i n the text. 1^-1X3 F R A C T I O N NUMBER 55 As shown i n the figure, COX-1 i n three d i f f e r e n t preparations existed i n the form of undefined aggregates with molecular weight ranging from 1 x 10 5 to greater than 1.5 x 10 6 daltons. However, Western b l o t assay revealed that a l l of the aggregates were derived from a basic unit of 60 KD. D. Assay of COX-1 by s o l i d phase Sandwich enzyme immunoassay In view of the f a c t that RP 215 recognized repeated epitopes of i t s complementary antigen, COX-1, a sandwich enzyme immunoassay using t h i s antibody was designed f o r quantitative determination of COX-1 i n d i f f e r e n t antigen preparations or i n human sera. The standard curve of t h i s enzyme immunoassay i s presented i n Figure 6. The s e n s i t i v i t y of t h i s enzyme immunoassay was determined to be 1 AU/ml. The i n t r a and inter-assay v a r i a t i o n i s between 5-15%. E. Characterization of rabbit antisera against COX-1 COX-1 protein spot was removed from two-dimensional gels of the a f f i n i t y p u r i f i e d COX-1 and used as an immunogen for the immunization of rabbits. The t i t e r s of rabbit antisera were determined by radioimmunobinding assay and by enzyme-linked immunosorbent assay using OC-3-VGH c e l l s coated on microwells. The r e s u l t s of t h i s immunoassay indicated that a f t e r successive immunizations, the t i t e r of 56 Figure 6 A standard curve of the solid-phase sandwich enzyme immunoassay for the quantitative determinations of COX-1 immunoactivity from 0C-3-VGH c e l l extract. The immunoactivity of COX-1 was expressed i n AU/ml as defined. Details of t h i s assay procedure were described i n the text. 58 rabbit antisera was determined to be 1:400 serum d i l u t i o n (as shown i n Figure 7). By western b l o t assay, the rabbit antisera were shown to react with a protein band of 60 KD molecular weight which was i d e n t i c a l to that of RP 215 monoclonal antibody (Figure 2). 59 Figure 7 Determination of t i t e r of rabbit anti-COX-1 by ELISA method as described i n the text, where ( £ ) and ( A ) indicate rabbit antiserum (after the t h i r d immunization) and normal rabbit serum, respectively. 200 400 800 1000 1200 1600 D I L U T I O N F A C T O R 61 X. Discussion During the l a s t decade, numerous investigations have been performed i n the search f o r " i d e a l " tumor markers which allow d i f f e r e n t i a t i o n between the normal and neoplastic conditions. This tumor marker should be highly s p e c i f i c to the tumor c e l l s and at the same time correlate with the prognosis and stage of the malignancy. Despite the continuing development of new therapeutic modalities, the most important factor influencing the prognosis of female g e n i t a l cancer i s early diagnosis. Early diagnosis of ovarian cancers i s d i f f i c u l t , mainly because of the lack of r e l i a b l e and s p e c i f i c tumor markers (Introna, H at a l , 1985). Currently, available cancer treatments such as radiotherapy and chemotherapy are often hampered by t h e i r apparent lack of tumor s p e c i f i c i t y and low therap e u t i c s / t o x i c i t y r a t i o s . Therefore, the development of such a marker f o r ovarian cancer i s es s e n t i a l as there appears to be l i t t l e chance of early diagonosis without such a diagnostic a i d . The rationale and importance of developing r e l i a b l e tumor markers f o r female g e n i t a l cancer, however, are not li m i t e d to early diagnosis of cancer. Such markers would enable gynecologic oncologists to follow-up patients a f t e r treatment and also to detect the recurrence at an early stage so that an additional or a new treatment modality 62 could be i n i t i a t e d . Development of a reactive antibody conjugated with a tracer substance to attack a cancer antigen could greatly enhance the accurate i d e n t i f i c a t i o n of tumor c e l l s that have spread beyond the o r i g i n a l s i t e of the cancers. Ultimately, chemotherapeutic or radiotherapeutic agents could be conjugated to these antibodies, enabling the physician to achieve s e l e c t i v e therapeutic eradication of cancer c e l l s while sparing normal t i s s u e s . As ovarian e p i t h e l i a l cancers usually produce no symptoms p r i o r to generalized intraabdominal dissemination of the disease and since there are no diagnostic screening tests that allow early diagnosis, i t could be useful i f a serum t e s t f o r tumor marker could be u t i l i z e d f o r early diagosis. Unfortunately, currently available tumor markers for ovarian cancer are neither s e n s i t i v e nor s p e c i f i c enough to enable early diagnosis. Development of the new marker i n the future would be b e n e f i c i a l f o r the c l i n i c i a n to diagnose cancers e a r l i e r and get better prognosis. With the development of hybridoma technology of monoclonal antibodies, RP 215 was generated and shown to be highly s p e c i f i c to sing l e epitopes of tumor-associated antigen COX-1. Monoclonal antibodies secreted by hybrid clones were i n i t i a l l y screened simultaneously by ELISA and i n d i r e c t immunofluorescent assay f o r t h e i r respective s p e c i f i c i t y to 63 cultured 0G-3-VGH c e l l s . The tissue s p e c i f i c i t y of the generated antibodies was examined by the quantitative ti s s u e adsorption experiment and immunohistochemical study. Among these monoclonal antibodies, RP 215 demonstrated the highest degree of s p e c i f i c i t y to OC-3-VGH tumor c e l l s and showed no cross r e a c t i v i t y to normal human tissues including l i v e r , t e s t i s , kidney, heart, muscle, and brain homogenate (Figure 1-1) and to tissue sections of vaginal wall, cervix, f a l l o p i a n tube, ovary, colon and small i n t e s t i n e , as resulted by the immunofluororescent method. In addition, RP 215 cross-reacted with some of the human gynecological cancer c e l l l i n e s employed i n t h i s study, including that of ME-180 c e l l s which are of c e r v i c a l e p i t h e l i a l o r i g i n (Table 4). Therefore i t may be reasonable to suggest that COX-1 may be a tumor-associated antigen derived from cancers of either ovarian or c e r v i c a l o r i g i n s . COX-1 cannot be detected i n the female serum during the p r o l i f e r a t i v e phase of the menstrual cycle which was assayed from 10 patients' samples c o l l e c t e d by Dr. Basal Ho Yuan i n Grace Hospital, Vancouver. The tumor-associated antigen, COX-1 was p u r i f i e d to homogeneity by using an immunoaffinity chromatographic procedure from the cultured shed medium or from the cultured tumor c e l l extract. COX-l appeared to e x i s t i n the state of aggregates i n the shed medium or i n tumor c e l l extract. However, a f t e r being p u r i f i e d from a f f i n i t y column by a low 64 pH buffer i n 50 mM glycine - HC1, pH 2.2, following ne u t r a l i z a t i o n , COX-1 continues to e x i s t as an undefined aggregate i n PBS. This was c l e a r l y demonstrated by a sepharyl S-300 gel f i l t r a t i o n chromatography. Following immunoaffinity p u r i f i c a t i o n , COX-1 retained immunoactivity to RP 215 when analyzed by dot bl o t immunoassay or Western b l o t assay. The aggregate form of COX-1 was unstable at temperatures above 50°C and se n s i t i v e to t r y p s i n (Figure 4) and other proteases. However, treatment of COX-1 with NaI0 4 and several carbohydrate-digestive enzymes including neuraminidase, beta-galactosidase and fucosidase d i d not a f f e c t the immunoactivity of COX-1. This c l e a r l y suggested that the carbohydrate moiety of COX-1 may not be es s e n t i a l f o r the binding between RP 215 and t h i s antigen. Rabbit antisera raised against p u r i f i e d COX-1 (50 ug) were shown to exhibit s i m i l a r immunospecificity to that of RP 215 monoclonal antibody. The polyclonal antisera might prove to be useful f o r immunoassay and immunohistological studies of t h i s tumor-associated antigen. A solid-phase sandwich enzyme immunoassay using RP 215 monoclonal antibody would enable us to detect the native form of COX-1 eithe r i n cultured shed medium of tumor c e l l s or i n sera of patients with ovarian or c e r v i c a l cancer. This enzyme immunoassay k i t might prove to be b e n e f i c i a l f o r 65 the diagnosis and monitoring of patients with ovarian or c e r v i c a l cancers. In order to explore the potential u t i l i t y of COX-1 enzyme immunoassay k i t f o r the diagnosis and monitoring of patients with c e r v i c a l and ovarian cancers, multi-center coll a b o r a t i v e studies are being conducted. Preliminary c l i n i c a l evaluation data were available from the following sources: (1) Department of Obstetrics and Gynecology Veterans General Hospital, T a i p e i , Taiwan, China (2) Department of Obstetrics and Gynecology Veterans General Hospital, Taichung, Taiwan, China (3) Hu-Bei Medical University , Wu Han, Hu-Bei, China Typical r e s u l t s are presented i n Table 6, Table 7, and Table 8. Due to the lack of absolute COX-l antigen absolute standards, the serum l e v e l s of COX-l among normal ind i v i d u a l s varied from the study sources. However, the general conclusions i n regard to the performance of t h i s immunoassay k i t were quite s i m i l a r . As shown i n Table 6, which reports data from VGH, Tai p e i , patients with ovarian cancer had higher l e v e l s of COX-l than those with benign tumor or p e l v i c mass. Serum COX-l l e v e l s among those patients with c e r v i c a l carcinoma seemed to be correlated with t h e i r respective disease stages. As many as 60-80% of patients with c e r v i c a l or ovarian cancers had 66 Table 6 Preliminary C l i n i c a l Evaluation of COX-1 among Patients with Ovarian, Ce r v i c a l Cancers, Benign Tumors or Normal Health Subjects** Normal Ovarian Disease Cervical Carcinoma Control D C * O T * PM*" Stages I II III Cases 130 20 19 8 25 20 5 Mean 29.4 82. 0 31. 4 45. 4 72.0 87.2 88.8 S.D. 19.4 34. 3 13. 1 25. 3 25.9 37.0 35.3 % P o s i t i v e * * * 80 5 13 64 80 80 Normal control vs. OC p<0.001 OC vs. OT p<0.001 OT non-significant OC vs. PM p<0.01 PM p>0.05 OT VS. PM p>0.05 vs. Cervical Carcinoma I p<0.001 I vs. II p>0.1 II p<0.001 I vs. I l l p>0.2 III p<0.001 * OC: Ovarian Cancer, AU/ml OT: Ovarian Tumor PM: Pe l v i c Mass ** data provided by Dr. H. T. Ng, Dept. of Obstetrics & Gynecology, Veterans General Hospital, T a i p e i , Taiwan. *** p o s i t i v e : > mean + 2SD (95% Confidence) 67 T a b l e 7 Serum l e v e l s of COX-1 of normal cases and patients with ovarian carcinoma, c e r v i c a l carcinoma, p e l v i c benign tumors and other tissue-originated tumors Cases Mean Values (Units/ml i n serum) S.D. Normal Control 10 2.1 1.51 Ovarian Carcinoma 18 43.8 7.90 Cerv i c a l Carcinoma 20 37.5 6.20 Pe l v i c benign tumors 4 21.3 5.36 Other Tissue-originated Carcinoma 10 8.08 5.33 Table 8 Comparision of serum COX-l le v e l s both i n pre-operative and post-operative cases for patients with ovarian carcinoma Pre--operative Post-operative ( 0 days 7 days) Number of cases 18 7 7 Mean Values (units/ml i n serum) 43.8 47.3 4.6 S.D. 7.9 6.88 2.49 P <0.001 C l i n i c a l data provided by Dr. E. Huang, Dept. of Obstetrics & Gynecology, Hu-Bei Medical University, Hu-Bei, China. 68 serum COX-1 l e v e l s s i g n i f i c a n t l y higher than those of the normal indiv i d u a l s ( > mean+ 2 S.D.)- S i m i l a r l y , the study from Hu-Bei Medical University seemed to suggest that serum COX-l le v e l s of those patients with c e r v i c a l or ovarian cancer were higher than those with other cancers or benign tumors (Tables 7,8). Furthermore, following s u r g i c a l removal of ovarian tumors, serum COX-l le v e l s appeared to decrease s i g n i f i c a n t l y 7 days a f t e r operation. Therefore, serum COX-1 le v e l s appeared to respond to the tumor burden of those cancer patients. Similar to other established tumor markers (CA 125, CA 19-9, NB/70K e t c . ) , the majority of patients with ovarian cancers showed s i g n i f i c a n t l y elevated l e v e l s of COx-l when compared with those of the normal control and those with endometriosis and p e l v i c benign tumor. In contrast to CA 125 and CA 19-9 which were not suitable tumor markers to monitor patients with c e r v i c a l carcinoma, COX-l could also serve as a se n s i t i v e marker for t h i s type of cancer. On the other hand, those patients with c e r v i c a l carcinoma could be monitored by a tumor marker derived from squamous c e l l carcinoma, SCC-antigen (Keto H. et al,1984; Maruo, T. et al,1985). COX-l seemed to have a comparable degree of s e n s i t i v i t y to that of SCC-antigen i n terms of the monitoring of patients with c e r v i c a l carcinoma as indicated i n Table 6. 69 Although serum COX-1 l e v e l s are less well correlated with the stage progression, the s i g n i f i c a n t l y elevated COX-1 l e v e l s could be r e a d i l y detected among those patients even with stage I of ovarian or c e r v i c a l carcinoma. The serum l e v e l s of COX-1 were also well correlated with conditions of c l i n i c a l treatments f o r those patients with ovarian carcinoma. This i s c l e a r l y demonstrated i n Table 8. Generally speaking, there was a s i g n i f i c a n t decrease i n serum COX-1 l e v e l s f o r those patients who had p r i o r s u r g i c a l removal of tumors seven days i n advance and those who underwent radio- or chemotherapy. 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