1501 A Novel Tumor-Associated Antigen Expressed in Human Uterine and Ovarian Carcinomas Kenzo Sonoda, M.D.’ Manabu Nakashima, M.D.’ Tsunehisa Kaku, M.D.’ Toshiharu Kamura, M.D.‘ Hitoo Nakano, M.D? Takeshi Watanabe, M.D.’ ’ Department of Molecular Immunology, Medical Institute of Bioregulation, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Department of Gynecology and Obstetrics, Faculty of Medicine, Kyiishu University, Fukuoka. Japan. The authors thank Dr. Masazumi Tsuneyoshi for providing paraffin embedded tissue blocks; Dr. Zenon Steplewski for thi: GA 733 MoAb; Dr. Akira Yachi for the YH 206 MoAb; Dr. Keigo Endo for the CA .I25 MoAb; Dr. Osamu Hayakawa for the CAC-1 cell; Dr. Masaru Sakamoto for the TMCC cell; Dr. Yoshihiro Kikuchi for the MH, KF, HRA, and KK cells; Dr. Masato Nishida for the HAG-2 cell; and the National Institute of Hygienic Sciences for the MCAS cell, Kuramochi, PA-I, TYK-nu, and BeWo cells. Address for reprints: Takisshi Watanabe, M.D., Department of Molecular Immunology, Medical institute of Bioregulation, Kyushu University 60, Maidashi 3-1 -1, Higashi-ltu, Fukuoka, 812-82, Japan. Received November 28,1995: accepted January 2, 1996. 0 1996 American Cancer Society BACKGROUND. A large number of monoclonal antibodies (MoAhs) against human tumor cells have been generated and it has been shown that these MoAbs are useful tools in the diagnosis and treatment of cancer patients, as well as in the basic investigation of the oncogenesis and characterization of cancer cells. METHODS. The 22-1-1 MoAb was established by cell fusion between mouse rnyeloma cells and spleen cells derived from mice immunized with the human uterine cervical adenocarcinoma cell line, SiSo. The tissue distribution and biologic characteristics of the 22-1-1 antigen (Ag) were examined. RESULTS. The 22-1-1 Ag was distinct from the known tumor-associated antigens such as YH 206, GA 733, CA 125, carcinoembryonic antigen, and sialyl Le”molecules in an expression pattern in human tumor cell lines. An immunohistochemical study revealed that 22-1-1 Ag was expressed in 87.5% of uterine cervical adenocarcinomas, 66% of uterine endometrial adenocarcinomas, and 58.8% of ovarian carcinomas. Moreover, 22-1-1 Ag was detected in 87.7% of uterine cervical squamous cell carcinomas; however, it was not detected in normal uterine cervical or ovarian tissues, except in uterine endometrial glands, in which its expression was observed at low levels. The 22-1-1 Ag was secreted into cell culture supernatant fluids and was also detected in the vaginal discharges of uterine cervical carcinoma patients. The antigenic epitope of 22-1-1 Ag was shown to be a protein with a molecular weight of 78 kilodaltons using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. CONCLUSIONS. The 22-1-1 MoAb reactive to a novel tumor-associated antigen was generated. This Ag was expressed in cancer cells derived mainly from the uterus and ovary. Moreover, 22-1-1 Ag was secreted in the vaginal discharges of uterine cervical carcinoma patients. 22-1-1 MoAb is a potential tool for the study of oncogenesis and the management of cancer patients. Cancer 1996; 721501-9. 0 1996 American Cancer Society. KEYWORDS monoclonal antibody, tumor-associated antigen, uterine carcinoma, ovarian carcinoma, flow cytometric analysis, irnmunohistochemical examination. T o date, many monoclonal antibodies (MoAbs) have been generated in the field of cancer research. Some MoAbs have been used to evaluatle the therapeutic effects of various treatments on cancer patient^.'-^ Other MoAbs have been applied to basic research to examine the growth characterization, immunogenicity, and physiology of tumor These investigations have shown that MoAbs are useful tools and the analysis of antigens detected by such MoAbs is important in the research of oncogenesis and the characteristics of cancer cells. Adenocarcinoma of the uterine cervix accounts for 5-20% of cervical cancers, and its incidence has recently been reported to be However, the carcinogenesis and biologic characteristics of this adenocarcinoma still remain unclear. Moreover, this type of cancer shows low sensitivity against radiation and 1502 CANCER April 15,1996 I Volume 77 / Number 8 chemotherapy in comparison with squamous cell carcinoma of the uterine c e ~ ~ i x . ’ Previously, ”’~ we established a new human cell line, SiSo, from a patient afflicted with uterine cervical adenocarcinoma to study the biologic characteristics of this d i ~ o r d e r .In ’ ~this report, we present a new MoAb, 22-1-1, against a novel tumor-associated antigen by immunizing mice with SiSo cell line. We investigated the tissue distribution and biologic property of 22-1-1 Ag and compared its characteristics with other tumor-associated antigens such as CA 125, CA 19-9, and carcinoembryonic antigen (CEA) using immunohistochemical analysis. MATERIALS AND METHODS Cell Lines The human tumor cell lines used in this study (Table 1) and a mouse myeloma cell line, X63. Ag8. 653, were maintained in a complete culture medium, RPMI- 1640, with fetal calf serum (Whittaker Bioproduct, Walkersville, MD) at 10% volume (of solute) per volume (of solvent) containing L-glutamine (1 rnM; Flow Laboratory, Irvine, Scotland), ,f?-mercaptoethanol (5 x Gibco Laboratories, Grand Island, NY), N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (Hepes) (pH 7.2) (10 mM; Gibco), nonessential amino acids (0.1 mM; Gibco), and sodium pyruvate (1 mM; Gibco). Immunization and Cell Fusion We previously established the SiSo cell line from a uterine cervical adenocarcinoma ~ a t i e n t . Balb/c ’~ mice (6 weeks old and female) were immunized subcutaneously with 1 x lo6 SiSo cells immersed with 0.5 mL of a complete adjuvant, Titer Max (CytRx, Norcross, GA). After 2 weeks, 1 x lo6SiSo cells mixed with incomplete Freund adjuvant (Organon Teknika, Turnhout, Belgium) were injected intraperitoneally for the second immunization. For a boost, sonicated 1 x lo6 SiSo cells in 1 mL phosphate-buffered saline (PBS) (pH 7) were used. Four days after the boost, spleen cells were fused with X63. Ag8. 653, at the ratio of 5:l (spleen cells:X63), using polyethylene glycol 4000. For monoclonal antibody screening, SiSo and other human tumor cell lines were stained by indirect immunofluorescence with the culture supernatant fluids of growing hybridoma colonies. Positive hybridomas were subcloned by limiting dilution. Supernatant fluids from cloned hybridomas were screened by indirect immunofluorescence for binding to uterine or ovarian carcinoma but not to other organ tumor cell lines. One positive clone was obtained and termed as 22-1-1. 22-1-1 MoAb was defined as an IgM class antibody. lrnmunofluorescence and Flow Cytometry The 22- 1-1Ag distribution on tumor cell lines was investigated by flow cytometric analysis. Briefly, each tumor cell TABLE 1 Expression of 22-1-1 Antigen on Human Tumor Cell Lines Cell line Pathologic diagnosis SiSo TMCC CAC-1 HeLa MH Kuramochi MCAS KF HRA KK HAC-2 PA-1 TK-nu BeWo K562 Daudi CCRF CEM MM-8 MM-28 MM-33.1 KB HEp-2 PC-10 GoTo ZR-75- 1 PC-9 A-549 MKN-45 Kato 111 Panc-1 Chang liver H1 CoLo 320DM CoLo 699 LOVO HT-1197 Uterine cervical adenocarcinonia Uterine cervical adenocarcinoma Uterine cervical adenocarcinoma Uterine cervical adenocarcinoma Ovarian mucinous adenocarcinoma Ovarian mucinous adenocarcinoma Ovarian mucinous adenocarcinonia Ovarian serous adenocarcinoma Ovarian serous adenocarcinoma Ovarian clear cell carcinoma Ovarian clear cell carcinoma Ovarian undifferentiated carcinoma Ovarian undifferentiated carcinoma Choriocarcinoma Erythroid leukemia Burkitt lymphoma Lymphoblastic leukemia Malignant melanoma Malignant melanoma Malignant melanoma Oral epidermoid carcinoma Laryngeal epidermoid carcinoma Lung squamous cell carcinoma Adrenal neuroblastorna Breast cancer Lung adenocarrinoma Lung adenocarcinonia Gastric cancer Gastric cancer Pancreatic cancer Liver cell Cholangioma Colon cancer Colon cancer Colon cancer Bladder cancer 22-1-1 A$ t:positive, -: negative. “The 2-1-1 Ag expression on tumor cell lines was detected by flow cylornetric analysis was harvested upon mild trypsinization, treated by 0.25% tqpsin with 0.5 mM ethylene diamine tetra acetic acid (EDTA),and 1 x lo5 cells were suspended in a cell-staining buffer PBS containing 0.5% bovine serum albumin and 0.05% NaN, with 10% goat serum for 20 minutes. The cells were then incubated with 100 yL of 22-1-1 hybridoma culture supernatant fluid on ice for 45 minutes. After washing 3 times, the cells were incubated with fluorescein isothiocyanate-conjugated goat antimouse IgM (Cappel Laboratories, West Chester, PA) antibody on ice for 45 minutes. The cells were washed 3 times with the staining buffer and a flow cytometric analysis was performed. At the same time, the reactivities of YH 206 MoAb,“ GA 733 M o A ~ , ‘ ~and , ’ ~CA 125 MoAblg against the cell lines were examined. Stained cells were analyzed A Novel Human Tumor-Associated Antigen/Sonoda et al. 1503 TABLE 2 22-1-1 Monoclonal Antibody Reactivity against Neoplasms in Female Genital Organs Pathologic diagnosis No. of cases IJterine cervixh Squamous dysplasia Squamous cell carcinoma in situ Microinvasive squamous cell carcinoma Invasive squamous cell carcinoma lnvasive adenocarcinoma 10 4 2 57 Uterine corpus Endometrial adenocarcinoma FIGURE 1. Adenocarciiioma of the uterine cervix. The 22-1-1 Ag existed both in the cytoplasm and on the membrane of cancer cells. Moreover, 22-1-1 Ag was detected in the glandular lumen of the tumor cells (magnification XIOO). t)y the Coulter Epics flowcytometry Epics Elite (Coulter, Iiialeah, FL). MoAb YH 206, GA 733, and CL4 125 were provided by Dr. Akira Yachi (Sapporo IJniversity School o f Medicine, Sapporo, Japan), Dr. Tenon Steplewski (The Wistar Institute. Philadelphia, PA), and Dr. Keigo Endo ( ( ;unnia Ilniversity School of Medicine, Gunma, Japan), respt:ctively. lrnrnunohistochemical Examination For immunohistochemical analysis, streptoavidin-biotin inethodology (IIistofine SAB-PO Kit; Nichirei, Tokyo, Japan) was used for formalin fixed and paraffin embedded ~pecin~ens.2"'The 22-1-1 Ag was localized with a 1 2 0 dilution of hybridoma culture supernatant fluid. Immunohistoclicniical analysis of other tumor-associated antigens, iricluding CA 125, C A 19-9 and CEA, was also performed. (:A 19-9 and CEA MoAbs were purchased from Nichirei (Tokyo, Japan). Paraffin embedded tissue blocks of nongynecologic cancer were provided by Dr. Masazumi Tsuneyoshi (Kyushu University, Fukuoka, Japan). Tissue sections with less than 5% reactive tumor cells were considcrcd negative, and those with more than 5% reactive cells was defined as positive. Chernilurninescent Western Dot Blotting I o tlctcct secreted 22-1-1 antigen in cell culture supernatant fluid, chemiluminescent Western dot blotting was performed. Briefly, each sample was applied to a nitroceliulosc~filter by a blotting apparatus (Rio-Rad, Richmond, (;A). The sample was fixed on the filter by air-drying. After 3% hydrogen peroxide (H,O,) treatment to remove a n endogenous peroxidase activity (10 minutes at room temperature). the filter was soaked in 5% nonfat milk in TrisI~ Ovaryh Mucinous cyst adenocarcinoma Serous cyst adenocarcinorna Clear cell carcinoma Endometrioid adenocarcinonia Total of ova:ian carcinomas No. of positive caw' 0 10%)' 64 2 (50%) 2 (100%) 50 (87.7%) 56 (87.5%) 103 64! (66%) 7 5 1 3 4 4 4 17 1 10 (58.8%) For immunohistochemiral analysis, streploavidin-biotinmerhodology was tised for formalin tixed and paraffin embedded specimens.Tissue sections with greater than 5% reaclive cells were defined as positive. "Nonrancrrous legions of uterine cenir or ovary \ w e negative for 22-1-1 ~ g . ' Kuniber in parenrhesisis he percenrage of positive c a w buffered saline-Tween 20 (TBS-T) containing 5% normal goat serum (45 minutes at room temperature). The filter was incubated with 22-1-1 MoAh in TBS-I' containing 5% normal goat serum for one hour at room temperature. After washing 3 times with TBS-T, the filter was incubated for 1 hour with peroxidase conjugated goat antimouse IgM antibody (Cappel Laboratories, West Chester, PA). After washing 3 times with TBS-T, the filter was soaked with enhanced chemiluminescence kit substances (Ametsham, Buckinghamshire, United Kingdom) and exposed for 15 minutes. Radioimrnunoprecipitation To investigate the molecular weight of 22-1-1 Ag, metabolic-radioactive labeling method was performed. Briefly, SiSo, Panc- 1, and Co1.0 699 were collected by mild trypsinization. After washing twice with PBS, each tumor cell was resuspended at a concentration of 5 x 10"cells/mL in 3 mL of methionine-free HPMI 1640 (Gibco I.aboratories, Grand Island, NY) containing 10% fetal calf serum dialyzed against PBS. After adding 100 pCi of I"'SJmethionine, the cells were incubated for 16 hours at a humidified 37 "C, 5% C 0 2 incubator. Labeled cells were collected and washed twice with ice-cold PRS. Each labeled cell pellet was incubated with a cell lysis buffer (15 mM nonanoyl-n-methyl-glucamide [Mega-g], 150 mbl NaCI, 5 mM EDTA, SO mM l'ris-HC1 IpH 7.51, 0.5% noni- 1504 CANCER April 15,1996 / Volume 77 / Number 8 TABLE 3 22-1-1 Monoclonal Antibody Reactivity against Genital Organ Tissues of Healthy Females Tissue No. of cases No. of positive cases Uterine cervix Uterine endometrium Proliferative phase Secretory phase Atrophic endometrium Ovary 5 0 3 3 4 5 la 3a Id 0 Only endometrial gland cells were weakly stained. FIGURE 2. lmmunohistochemical analysis of the uterine cervix by 221-1 MoAb. Normal uterine cervical tissue is negative for 22-1-1 Ag A: squamous epithelium, B: cervical gland cells. (Original magnification X200). det P-40 [NP-401,and 2 mM phenyl methyl sulfonyl fluoride [PMSFI) for 15 minutes at 4 "C. The cell lysate was incubated with 22- 1- 1 MoAb, after which goat antimouse IgM antibody (Cappel Laboratories West Chester, PA) and, finally, protein A beads (Pierce, Rockford, IL) were added (1 hour at 4 "C, respectively). The eluate was electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced and nonreduced conditions. Enzyme Treatment Partial purified 22-1-1 Ag from culture supernatant fluid by ammonium precipitation was treated by trypsin (Difco Laboratories, Detroit, MI), neuraminidase (Sigma, St. Louis, MO), and 0-glycanase (Genzyme, Cambridge, MA). Briefly, 22-1-1 Ag was incubated with trypsin at concentrations from 0.01 to 10 mglmL (20 hours at room temperature). After incubation, 22- 1- 1 Ag was transferred onto nitrocellulose filters for Western blot analysis. 22- 1- 1 Ag was then treated by 0-glycanase (4 mUl100 pg protein of sample) in 10 mM calcium acetate, and 20 mM sodium cacodylate solution (pH 6.8) (20 hours at 37 "C). After, 22-1-1 Ag was treated by neuraminidase (20 mUlmL) in 0.1 M sodium acetate buffer (pH 5) (4 hours at 37 "C). SiSo cells were treated by neuraminidase (Sigma, St. Louis, MO), hyaluronidase (Sigma),tunicamycin (Sigma), and N-acetyl a - D galactosaminidase (Sigma), after which the expression of 22- 1-1Ag was analyzed by flow cytometry. Briefly, SiSo cells were treated by 10-1000 UlmL of hyaluronidase with 500 UlmL of aprotinin in HEPESbuffer saline (30 minutes at 37 "C). SiSo cells were then cultured with tunicamycin (0- 10 pg/mL, for 24 hours and 96 hours). After SiSo cells were incubated with 5 mM of N-acetyl a-D galactosaminidase in culture medium (36 hours at 37 "C). Then, 1 x 10' SiSo cells were treated by neuraminidase (100 mU/mL) in HEPES-buffer saline (20 mM of HEPES, and 140 mM of NaCl [pH 71) at 37 "C for 30 minutes. RESULTS 22-1-1 Ag Distribution on Human Tumor Cell lines The 22-1-1 MoAb reacted with tumor cell lines derived from uterine cervical adenocarcinoma and ovarian adenocarcinoma, such as SiSo, TMCC, MH, Kuramochi, KF, and HRA. However, it did not react with any tested tumor cell lines that were derived from other organs (Table 1). In comparison with MoAbs reactive to other tumor-associated antigens, such as YH 206, GA 733, and CA 125, the distribution pattern of 22-1-1 Ag was significantly different (data not shown). Additionally, Kato 111, which expressed CEA and sialyl Le" molecules, was negative for 22-1-1 Ag. Therefore, the 22-1-1 Ag was distinct from YH 206, GA 733, CA 125, CEA, and sialyl Le" molecules. Tissue Distribution of 22-1 -1 Antigen Immunohistochemical analysis with 22-1- 1 MoAb was performed using paraffin embedded specimens. 22-1- 1 Ag existed both in the cytoplasm and on the membrane of the adenocarcinoma cells derived from the uterine cervix. Moreover, 22-1-1 Ag was detected in the glandular lumen of the cancer cells, indicating that 22-1-1 Ag was also secreted (Fig. 1). The 22-1-1 MoAb reacted with 87.5% of the cases with uterine cervical adenocarcinomas, 66% of the cases with uterine endometrial adenocarcinoma, and 58.8% of the cases with ovarian carcinoma (Table 2). The 22-11 MoAb also reacted with adenoma malignum (highly differentiated adenocarcinoma) of the uterine cervix. With regard to ovarian carcinomas, 22-1-1 MoAb stained mucinous adenocarcinomas stronger than serous or endometrioid adenocarcinomas. 22- 1-1 Ag was detected not A Novel Human Tumor-Associated Antigen/Sonoda et al. 1505 TABLE 4 Reactivities of Various Monoclonal Antibodies against Uterine Carcinomas MoAb 22-1-1 0 125 CA 19-9 CFA Uterine cervical squamous cell carcinoma Uterine cervical Uterine endometrial adenocarcinoma adenocarcinoma 50/57 (87.7%)a 56/64 (87.5%) 27/40 (67.5%) 29/40 (72.5%) 36/40 (90%) 4/10 (40'%) 5/10 (50%) 6/10 (8O'?h) 681103 (66%) 30140 (75%) 19/40 (47.5%) 10/40 (25%) C U : carcinoembrponic antigen. Numbers of positive cases of numbers of total cases. Nuinbers in parentheses are percent positivity. a FIGURE 3. The secretion of 22-1-1 Ag to the cell culture supernatant fluid. The supernatant fluid of each cell line at the indicated initial cell number and culture period was applied to a nitrocellulose filter. Filters were incubated with 22-1-1 MoAb followed by peroxidase conjugate goat antimouse IgM. Detection of 22-1-1 Ag was performed with a chemiluminescent substance. only in adenocarcinomas of the female genital tract, but also in 87.7% of cases with uterine cervical squamous cell carcinoma. With regard to uterine cervical squamous cell neoplasms, 22-1-1 MoAb did not react with squamous dysplasia but it strongly stained the cytoplasm of microinvasive arid invasive carcinoma cells (Table 2). 22-11 MoAb reacted weakly with normal uterine endometrial gland cells. However, no reactivity was observed in the normal uterine cervical squamous epithelium, cervical gland cells (Fig. 2), or ovarian tissues (Table 3). The 22-1-1 MoAb reactivity was compared with that of CA 125, CA 19-9, and CEA MoAbs in uterine cancers (Table 4). 22-1-1 Ag was expressed in a comparatively broader and stronger manner in uterine cancers than were other tumor-associated antigens. The Secretion of 22-1-1 Ag to Culture Supernatant Fluid The culture supernatant fluids of SiSo, HeLa, and CAC-1 were examined in the secretions of 22-1-1 Ag (Fig. 3). 221-1 Ag was detected in the culture supernatant fluids of FIGURE 4. The 22-1-1 Ag was detected in the vaginal discharges iof uterine cervical cancer patients. The vaginal discharges were collected from healthy donors and cancer patients. Protein concentration of samples was determined by optical density at 280 nm and was adjusted to O.!% 100 p,L of each indicated diluted sample was used for Western blot anal!/sis. The vaginal discharge samples were collected from healthy donors (lanes 1 to 8), a uterine cervical squamous cell carcinoma patient (lane 9), and uterine cervical adenocarcinoma patients (lanes 10 and 11). Lanes 12 and 13 contain the culture supernatant fluids of SiSo cells and phosphate-buffered saline, respectively. FIGURE 5. The molecular size of 22-1-1 Ag was estimated. Each cell line was labeled metabolically with [35S]-methionine.22-1-1 Ag was immunoprecipitated and analyzed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reduced and nonreduced conditions. Cell line 1:SiSo; 2:Panc-1; 3:CoLo 699. SiSo cells that expressed 22-1-1 Ag and the amount of secreted 22-1-1 Ag was found to increase depending on the culture period and cell number. However, 22-1-1 Ag was not found in the supernatant fluids of HeLa and CAC.1 in which 22-1-1 Ag was negative. Moreover, 22- 1- 1 Ag was also detected in the vaginal discharges of uterine cervical cancer patients, but not in normal healthy donors (Fig. 4). Molecular Size of 22-1-1 Ag The molecular size of 22-1-1 Ag was assessed using SiSo cell lysates after [%I -metabolic labeling. Molecules of apparently 78 kilodaltons in weight were detected in SiSo cell lysates under both reduced and nonreduced conditions of sodium dodecyl sulfate-polyacrylamide gel elec- 1506 CANCER April 15,1996 / Volume 77 / Number 8 TABLE 5 Sensitivity of 22-1-1 Antigen to the Treatment by Various Enzymes Enzyme treatment Condition for reaction Substrates Sensitivity of 22-1-1 antigen Trypsid Hyaluronidaseb Tunicamycinb O-glycanasea N-acetyl IY-Dgalactosaminidaseb Neuraminidase' 0.01-10 mglmL, 20 hr, RT 10-1000 UlmL, 30 min, 37 'C 10 mglmL, 24-96 hr, 37 'C 4 mu,20 hr, 37 'C 5 mM,36 hr,37 'C 20-100 mUlmL, 37 'C Protein N-linked glycan K-linked glycan O-linked glycan O-linked glycan Sialic acid Sensitive Resistant Resistant Resistant Resistant Resistant hr: hour; min: minute; R T room temperature. Partially purified 22-1-1 antigen from culture supernatant fluid of SiSo cells was treated with each enzyme and Western dot blotting was performed using 22-1-1 MoAb. SiSo cells were incubated with each enzyme and the cell surface expression of 22-1-1 antigen was then analyzed by flow cytomey. 'The partially purified 22-1-1 Ag was treated with neuraminidase for 4 hours and analyzed by Western dot blotong. SiSo cells were also treated with neuraminidase for 30 minutes and the evpression of 22-1-1 Ag on the cell surface was analyzed by flow cytornetiy. a trophoresis; however, they were not seen in lysates of Panc-1 and CoLo 699 cells that were not positive for 221-1 Ag (Fig. 5). Moreover, affinity purified 22-1-1 Ag from SiSo cell culture supernatant fluids was also shown to have the same molecular weight of 78 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown). Sensitivity of 22-1-1 Ag Against Enzyme Treatment The sensitivity of 22-1-1 Ag against various enzymes was investigated (Table 5). 22-1-1 Ag was shown to be sensitive to trypsin treatment, because 22-1-1 antigenicity was scarcely detected after trypsin digestion in a concentration-dependent manner (data not shown). Conversely, 22-1-1 Ag was resistant to treatment with hyaluronidase, tunicamycin, O-glycanase, N-acetyl a - D galactosaminidase, and neuraminidase. DISCUSSION To date, many MoAbs have been generated in the field of cancer research and the utility of these MoAbs has been extensively investigated. MoAbs such as CA 125, CA 19-9, CEA, and alpha-fetoprotein have been used to detect tumor-associated antigens in the serum of cancer patients after treatment. Radioisotope-conjugated MoAbs have been employed to detect tumor tissues of primary sites and m e t a s t a s e ~ . * ~ ~~ A-number of cancer therapies using MoAb specific for tumor-associated antigens have been attempted. In addition to the direct effects of MoAbs by antibody-dependent cellular cytotoxicity and complement-dependent cellular c y t o t o x i ~ i t y , ~anticancer ~-~~ drugs- or radionuclides- conjugated MoAbs have been applied in the treatment of ~ a n c e r . ~Moreover, *-~~ several MoAbs have been manufactured for immunohistologic discrimination of benign versus malignant cells in histologic and cytologic preparation^.^^-^' These MoAbs also have been used to examine the origin, growth profile, and function of tumor In the case of gynecologic neoplasms, several MoAbs have been reported to be useful. B72.3 and Ca 1 MoAbs were reactive with both endometrial hyperplasias and carcinoma^.^'^^^ CA 1, HMFG 1, and HMFG 2 MoAbs were found to be most reactive with immature metaplasia as opposed to squamous cell carcinoma^.^^ CEA MoAb has been shown to be of value both in the discrimination of ovarian primary adenocarcinomas from metastatic adenocarcinomas of colonic origin and in the differentiation of endocervical versus endometrial adenocarcinoOC 125 MoAb was reported to recognize a majority of serous, clear cell, endometrioid, and undifferentiated ovarian tumors, but not mucinous, sex cord, or germ cell t ~ m o r s .MSN-1 ~ ~ . ~MoAb ~ has been shown to be useful in confirming endometrioid or clear cell differentiation in ovarian and endometrial tumors and in distinguishing between atypical endometrial hyperplasias and those without a t ~ p i a . ~1C5 ' MoAb, which reacted with various adenocarcinomas derived from the female genital tract but not with squamous cell carcinomas, was reported to be an improved tool for the detection of adenocarcinomas of the uterine cervix through tissue imprints and cervical smears.48 We produced 22-1-1 MoAb from mice immunized with SiSo cells derived from uterine cervical adenocarcinoma. Although 22- 1- 1 MoAb reacted weakly with normal glandular cells of the uterine endometrium, it was not detected in the normal uterine cervix or ovarian tissues. On the contrary, 22-1-1 MoAb strongly stained uterine cervical adenocarcinomas (87.5%), uterine endometrial adenocarcinomas (66%), and ovarian adenocarcinomas (58.8%).Moreover, 22-1-1 MoAb was shown to react with squamous cell carcinomas of the uterine cervix (87.7%). In addition, there is a possibility that 22-1-1 Ag expression A Novel Human Tumor-Associated Antigen/Sonoda et al. 1507 TABLE 6 Reativities of the Combination of 22-1-1 MoAb with Any One of Either CA 125, CA 19-9, or CFA MoAbs - MOW Uterine cervical squamous cell carcinoma Uterine cervical adenocarcinoma Uterine endometrial adenocarcinoma 22-1-1 \nth CA 125 22-1-1 mth CA 19-9 22-L-1 wth CM 9\10 9/10 (90%) lO/lO (100%) 38140 (95%) 39/40 (97.5%) 38/40 (95%) 35/40 (87 5%) 28/40 (70%) 23/40 (57 5%) - - CEA carcinoenibryonic antigen Numbers of positive cases of numbers of total cases Numbers in parenthesis are percent posltivlty is related to a neoplastic progression, because 22-1-1 Ag is detectable in rnicroinvasive and invasive squamous cell carcinomas but not in dysplasia of the uterine cervix (Table 2). In nongynecologic cancers, 22-1-1 Ag was detected in esophageal squamous cell carcinomas (two positive cases of two total cases), gastric adenocarcinomas (three positive cases of three total cases), colon adenocarcinomas (three positive cases of three total cases), and pancreatic adenocarciriomas (two positive cases of two total cases), indicatiq that 22-1-1 Ag exists widely in various cancer tissues. We compared the tissue distribution of 22-1-1, CA 125, CA 19-9, and CEA Ag in uterine cancers. As reported previo~sly,"".~~~' CA 125 MoAb showed a significant reaction with uterine adenocarcinomas (67.5-75%), but hardly any with squamous cell carcinomas (as shown in Table 4). CA 19-9 MoAb was reported to react with 80.392.5% of uterine endometrial adenocarcinomas."~5' However, in OUT study, CA 19-9 Ag was detected in only 47.5% of endometrial adenocarcinomas, whereas 22- 1- 1 Ag existed in 87% of uterine cervical carcinomas and in 66% of endometrial adenocarcinomas. Therefore, 22- 1-1 MoAb appears to react with uterine cancer cells more broadly than CA 1;!5 and CA 19-9 MoAbs. CEA MoAb has been shown to he of use in the differentiation of endocervical versus endomei rial adenocarcinomas, because CEA MoAb reacted with uterine cervical adenocarcinomas more strongly 1 han with endometrial adenocarcinoma^.^^-^^ Mle found this to be true in our study as well. (Table 4). With regard to squamous cell carcinomas, CEA Ag was detected in 80% of tumor tissues, with this reactivity being slightly higher than that of a previous report.53 In addition, the combination of 22-1-1 MoAb with any one of either CA, 125, CA 19-9, or CEA MoAb detected greater than 90% of uterine cervical squamous cell carcinomas (Table 6). The combination of 22-1-1 MoAb with CA 19-9 MoAb stained 97.5% of the cervical adenocarcinomas, and 22-1-1 MoAb combined with CA 125 MoAb reacted with 87 5% of endometrial adenocarcinomas. This data indicates that when 22-1-1 MoAb is combined with one of the other MoAbs, it can detect cancer cells more efficiently than any other MoAb alone. This might be of value in diagnosing uterine cancer tissues. In preliminary experiments, 22-1-1 Ag was detected in the vaginal discharges of uterine cervical cancer patients, but not in that of normal healthy donors (Fig. 41, indicating that 22-1-1 MoAb can be used in the diagnosis of uterine cancer with vaginal discharge. This clinical relevance of 22-1-1 MoAb is now under investigation. From flow cytometric analisis data, 22-1-1 Ag was shown to be distinct from YH 206, GA 733, CA 125, CEA, and sialyl Le" molecules. The tissue distribution of 221-1Ag was also different from those of CA 125, CA 199, and CEA antigens. Moreover, there is a possibility that 22-1-1 Ag expression is related to a neoplastic progression. 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