[CANCER RESEARCH 46, 4770-4775, September 1986] Two Novel Cell Surface Antigens on Small Cell Lung Carcinoma Defined by Mouse Monoclonal Antibodies NE-25 and PE-351 Takashi Takahashi, Ryuzo Ueda, Xiaoshi Song,2 Keiko Nishida, Masanori Shinzato, Reiko Namikawa, Yutaka Ariyoshi, Kazuo Ota, Kanefusa Kato, Toshiharu Nagatsu, Munehisa Imaizumi, Toshio Abe, and Toshitada Takahashi3 Departments of Thoracic Surgery ¡Ta.T., M. I., T. A.] and Biochemistry [T. N.], Nagoya University School of Medicine, Showa-ku, Nagoya 466; Laboratories of Chemotherapy and Immunology [R. U., X. S., K. !\t., To. T.] and Department of Internal Medicine [Y. A., K. O.J, Aichi Cancer Center, Chikusa-ku, Nagoya 464; Institute for Comprehensive Medical Science [M. S.J, Fujita-Gakuen Health University, Toyoake, Aichi 470-11; First Department of Pathology [R. N.], Aichi Medical University, Nagakute-cho, Aichi 480-11; and Department of Biochemistry [K. K.], Institute for Developmental Research, Aichi Prefectura! Colony, Kasugai, Aichi 480- 03, Japan ABSTRACT Two mouse monoclonal antibodies, NE-25 and PE-35, defining novel cell surface antigens of small cell lung carcinoma (SCLC) were produced. The molecular weight of NE-25 and PE-35 antigens estimated by ra- dioimmunoprecipitation was 25,000 and 35,000, respectively. NE-25 antigen was expressed on the majority of cell lines and tumor specimens of SCLC among lung carcinoma. These NE-25-positive cell lines showed typical growth morphology as SCLC classic lines and expressed high levels of neuroendocrine biomarkers, such as aromatic i - amino acid decarboxylase, while NE-25 antigen-negative lines lacked apparent neuroendocrine properties. This antigen was expressed also on a subset of neoplastic cells with (neuro)endocrine properties, including pulmonary carcinoid, and on various tumors of nervous tissues, such as neuroblastoma. Among the normal cells, Kulchitski cells of lung, thyroid gland, adrenal gland, Langerhans islet, and nervous tissues were positive. Thus, the expression of NE-25 antigen is closely associated with the neural and/or (neuro)endocrine differentiation state. On the contrary, PE-35 antigen was present on four major types of lung carcinomas as well as on squamous cell carcinoma and adenocarci- nomas of various tissues, but it was absent from nervous tissue tumors. Thus, PE-35 antibody showed a "pan-epithelial" reactivity. Analysis by NE-25 and PE-35 antibodies provided evidence for the heterogeneities of SCLC by demonstrating four surface phenotypes, with the NE-25+/PE-35* phenotype being most common. In addition, the results supported the current understanding that various histológica! types of lung carcinoma, including SCLC, are derived from a stem cell of the bronchial epithelium. genetic, and biochemical features among lung carcinomas (3- 5). Heterogeneity among SCLCs from different patients and even within an individual patient's tumors was reported to exist and has clinical importance (6-8). Therefore, MoAbs which may distinguish such heterogeneity would be of great value. To date, only a few MoAbs with the reactivity restricted to SCLC (9-14) have been developed, however. Huang et al. ( 15) reported that many MoAbs obtained from immunization with SCLC cell lines were directed against Lacto-7V-Fucopentaose III, which is not restricted to SCLC. In addition, lung carcinoma cell lines including SCLC as well as other carcinoma cell lines were reported to express blood group antigens, which are known to have strong immunogenicity (16). Since neuroblastoma cells rarely express blood group anti gens and have a surface protein distribution similar to SCLC more than to other major types of lung carcinoma in two- dimensional gel electrophoresis (17), not only SCLC cells, but also neuroblastoma cells were used in this study for immuni zation of mice to obtain MoAbs detecting SCLC-associated antigens. This paper presents a description of antigens detected by two MoAbs, NE-25 and PE-35. The expression of NE-25 antigen was closely associated with neural and/or (neuro)en- docrine differentiation properties, while PE-35 antigens were present on the majority of neoplastic cells and normal epithelial cells. INTRODUCTION With the recent advent of hybridoma techniques, the identi fication of antigens on tumor cells by MoAbs4 has contributed to better understanding of tumor biology in various human tumor systems, such as melanoma, renal cell carcinoma, and hematopoietic tumors ( 1). MoAbs also have demonstrated po tential usefulness in clinical applications, including the immu- nohistological classification of tumors, serum diagnosis, detec tion of métastasesby radioimaging, and use as therapeutic agents (2). SCLC has distinct clinical, morphological, biological, cyto- Received 4/10/86; accepted 5/23/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported in part by a Grant-in-Aid for the Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare; Grant-in-Aids for Cancer Research from the Ministries of Education, Science, and Culture and of Health and Welfare in Japan; and by a grant from the Cancer Research Institute, Inc., New York, NY. 2 Present address: Department of Immunology, Cancer Institute, Harbin Med ical College, Harbin, China. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: MoAb, monoclonal antibody; SCLC, small cell lung carcinoma; AADC, aromatic L-amino acid decarboxylase; NSE, neuron specific enolase; CK-BB, brain isozyme of creatine kinase; APUD, amine precur sor uptake and decarboxylation; M-MHA, mixed hemadsorption; ABC, avidin- biotin peroxidase complex. MATERIALS AND METHODS Cell Lines. All the cell lines derived from lung tumors and other malignancies were maintained in either RPMI-1640 or Eagle's minimal essential medium supplemented with streptomycin (100 fjg/ml), peni cillin (100 units/ml), 2 HIMglutamine, and 10% fetal bovine serum. SCLC-SA (18), SCLC-SM, and SCLC-MO were established from patients who were histologically diagnosed as having oat cell carcinoma of the lung at Aichi Cancer Center. SCLC cell lines used in this study were characterized by growth patterns and biochemical markers (Table 2). Based on the culture appearance by phase-contrast microscopy, they were subgrouped into 4 categories according to classification by Carney et al. (4). AADC (EC 4.1.1.28) was assayed based on the enzymatically formed dopamine by high-performance liquid chromatography with electrochemical detection as previously described ( 19). NSE, -,-subun¡t of enolase (EC 4.2.1.11), was measured using sandwich enzyme im- munoassay as previously described (20). The concentration of CK-BB (EC 2.7.3.2) was determined by sandwich immunoassay as described previously (21). Production of Hybridomas and Serological Procedure. BALB/c mice were immunized 3 times with either mixture of two SCLC cell lines (SCLC-SA and SCLC-SM) or cryopreserved neuroblastoma cells from a 3-yr-old female (No. 1134). Three days after the last immunization, spleen cells were obtained from the mice and fused with NS-1 myeloma cells by polyethylene glycol (M, 4000) as previously described (22). After 2-3 wk of culture in hypoxanthine, aminopterin, and thymidine, the reactivity of the supernatant was screened by M-MHA and immune 4770