30, 403-408 (1981)
Antigens Detected on Human Lung Carcinoma by Hybridoma Monoclonal Antibody’
KASAI, M.D.,’ ROMAINE E. SAXTON, PH.D., E. CARMACK HOLMES, MARTYN W. BLJRK, M.D., PH.D., AND DONALD L. MORTON, M.D.
Surgical Service, Veteran’s Administration Medical Center, Sepulveda, Division of Oncology, Department of Surgery, University of California Los Angeles, California 90024
California 91343, and School of Medicine,
Presented at the Annual Meeting of the Association for Academic Surgery, Birmingham, Alabama, November 5-8, 1980 Hybridoma monoclonal antibodies were used to identify tumor cell membrane antigens on a new human lung carcinoma cell line. Hybridomas were constructed by fusing S194 mouse myeloma cells with lymphocytes from Balb/C mice immunized by the human carcinoma cell line UCLA-SO-P3. Of 768 hybridoma cultures tested 40 secreted antibody reacting with these P3 tumor cells by an indirect lZ51-proteinA binding assay. Three produced antibody binding selectively to the P3 tumor cells, but not with a lymphoblastoid cell line from this same cancer patient. These 3 hybridomas were cloned by limiting dilution and antibody subclass was determined. The first monoclonal antibody reacted only with the P3 lung carcinoma and with one colon carcinoma line. The second cross-reacted with most human carcinomas tested including lung, colon, and breast carcinoma, but not with sarcomas, melanomas, embryonic cells, lymphoblastoid cells, or normal lymphocytes. The third reacted strongly with all the tumor cell lines tested except for sarcoma and with embryonic cells but not with lymphoblastoid cells or lymphocytes. These results show that monoclonal antibodies can be produced against a variety of different membrane antigens on this lung carcinoma cell Lineincluding several that may prove useful in diagnosis or treatment of human cancer.
been extensively studied with monoclonal antibodies. In recent years, hybridoma monoclonal In the present study, we have constructed antibodies have been used to identify cell lymphocyte-myeloma hybridomas that prosurface antigens on leukemias, melanomas, duced antibody against a human squamous and other human tumors [l, 3, 5, 9, 10, 17, cell carcinoma line. These hybridoma 181. In several cases, it was possible to monoclonal antibodies were tested for bindisolate and characterize unique tumor antiing to an autologous B-lymphoblastoid cell gens from these malignant cells [IO, 171. Although pulmonary carcinoma is one of line in order to exclude the cross-reactivity with HLA antigens. Three of these monothe most common human tumors and is the clonal antibodies show a unique pattern of subject of intensive research to identify reactivity against an antigenic determinant specific antigens expressed on lung cancer expressed on human tumor cells. cells [ 11, 141that will be useful in diagnosis and treatment , lung tumor cells have not INTRODUCTION
’ Supported in part by Medical Research Services, Veterans Administration and by USPHS Grant CA 12582, awarded by the National Cancer Institute (DHEW). * To whom requests for reprints should be sent: 54-140CHS, UCLA School of Medicine, Los Angeles, Calif. 90024.
Cell lines. The pulmonary tumor cell line, designated UCLA-SO-P3, hereafter called P3, is a squamous cell carcinoma of the lung derived from a 53-year-old white male. P3a is a metastatic lung carcinoma from the same patient. The autologous B-lympho-
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JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 4, APRIL 1981 TABLE 1
Immunization and fusion. Five IO-weekold Balb/C mice were immunized by intraperitoneal injection with 10’ P3 tumor cells 1 week apart. Three days after the last inocuCulture P3 cells” PL3 cells lation, sensitized spleen cells (5 x lo* cells) well No. Ratio (cpm) (cpm) were harvested, mixed with 1 x lo* S194 myeloma cells, and fused in 50% poly1:l 180 C7 5,195 4836 ethyleneglycol 1500 as described by Oi and 25: 1 169 D4 19,014 725 23,211 2753 8:1 166 E9 Herzenberg [ 121.The fused cells were trans6:1 171 C8 3,902 659 ferred to Costar 96-well microculture plates 167 F4 2,779 7126 1:3 (6 x lo5 cells/well) and cultured for 2 weeks 172 ES 1,195 801 1:l in RPM1 1640 with 10% FCS containing hypoxanthine (13.6 mg/liter), aminopterin a P3 or PL3 cells (2 x 105/50~1) incubated 30 min at 3PC, with hybridoma culture supematants, washed (0.176 mg/liter), and thymidine (3.88 mg/liter) three times, incubated with polyvalent antiglobulin (HAT medium) to select lymphocyte(1:50), washed three times, incubated with Pz5-protein myeloma hybrid cells. The hybrid cells were A(5 x 106cpm/pg, 2 x 105cpm/50 ~1). grown in culture medium containing hypoxanthine and thymidine (HT medium) for blastoid line (PL3) was established in cul- another week. After the antibody detection ture from the patient’s peripheral blood assay, the positive hybridomas were cloned lymphocytes by’ infection with EB virus. by the limiting dilution method. Lung carcinoma lines, UCLA-SO-PI, Antibody detection assay. An indirect -P4, -PS, -P6, and -W, were established in protein A assay was developed to detect our laboratory. Melanoma lines, MlO, M12, hybridoma antibody binding to the P3 tumor M14, MIS, M19, M20, M23, M25, and M29, cells similar to that described elsewhere were established in our laboratory as de- . In brief, the tumor target cells in susscribed previously . MDA-MB- 157, -23 1 pension or as monolayers were incubated and -734, hereafter called 157,231, and 734, with hybridoma culture fluids, then washed are breast carcinoma lines from other and incubated with rabbit anti-mouse globlaboratories . ulins (DAK0 1:50), washed again, and inHuman cells were grown in RPM1 1640 cubated with 1251-proteinA (2 x lo5 cpm/ (Gibco) with 10% fetal calf serum, penicillin 50 ~1). The protein A reagent (SPA) was (100 units/ml), and streptomycin (100 mgl labeled with 1251(2 x lo7 cpm/pg SpA) by liter). Sarcoma lines Sl and S3 [ 131, colon the vapor-phase chloramine-T method as we carcinoma line Don, and whole human described previously . embryonic cell WHE were established in RESULTS our laboratory. HT29 is a colon carcinoma line. B-Lymphoblastoid lines L14, L20, Identification of Antibody-Producing and SLl were established from M14, M20, Hybridomas and Sl patients’ peripheral blood lymphoAfter 2 weeks of HAT selection, culture cytes, respectively, by infection with EB supematants from the hybridoma primary virus in our laboratory. Six donors’ peripheral blood lympho- culture wells were screened for antibody cytes (PBL) (JK, SG, NB, BP, AK, DK) binding to P3 carcinoma monolayers by the indirect protein A assay using X-ray film were also obtained. S194 is a non-immunoglobulin-producing, bromouridine deoxy- autoradiography. Positive hybridomas were ribose (BUdR)-resistant myeloma cell line then tested again for antibody binding to P3 from Balb/C mice which was kindly provided tumor cells and to the autologous lymphoblastoid line PL3 using a suspension assay. by the Salk Institute, San Diego, California. HYBRIDOMAANTIBODYBINDINGTOLUNGCARCINOMA AND AUTOLOGOUS LYMPHOBLASTOID CELLS
KASAI ET AL.: MONOCLONAL
An example of the binding patterns of different hybridoma antibodies to the P3 tumor and paired nontumor PL3 cells is shown in Table 1. The hybridoma antibody 169D4 binds 25fold more strongly to P3 tumor cells than to autologous PL3 lymphoblasts. 18OC7and 172E5 bind nearly equally to P3 and PL3 cells. On the contrary, 167F4 binds preferentially with PL3. Hybridomas which produce antibodies binding more than five times higher to P3 than to the paired PL3 were then cloned as single cells by the limiting dilution method. Preparation
In order to obtain high-titer monoclonal antibodies, cloned hybridoma cells were inoculated intraperitoneally ( lo7 cells per mouse) into mineral-oil-primed Balbk mice. The ascites fluid was collected 7 days later after tumor inoculation. As shown in Fig. 1, the 169D4 hybridoma monoclonal antibody shows very strong specific binding to P3 cells even at a dilution of 1 to 10,000. Only at a 1:lO dilution is any binding to autologous lymphoblasts seen and this is probably due to low-titer mouse natural antibody in the ascites fluid. Irnmunoglobulin Antibodies
Subclass of Hybridoma
Table 2 shows the immunoglobulin subclass of 169D4,1721)5, and 179E3hybridoma antibodies. Class-specific rabbit anti-mouse immunoglobulin was used as second antibody and then lz51-protein A was added. An indirect three-step binding assay allowed
FIG. 1. Titration of ascites antibody from hybridoma 169D4 using 1Z51-proteinA binding assay. The monoclonal antibody was tested against P3(0) and PL3(0).
us to identify the immunoglobulin isotype. None of them showed the ability to bind protein A directly. 169D4 is IgM subclass, 172D5, and 179E3 are IgG,. Specificity of Hybridoma Antibodies
Binding of hybridoma antibodies to surface components of various human cells was assessed using the indirect Y-SPA binding assay. Target cells include human lung carcinomas, colon carcinomas, breast carcinomas, melanomas, sarcomas, embryonic cells, B-lymphoblastoid cells, and peripheral lymphocytes. As shown in Fig. 2, antibody from 169D4 binds only to the P3 cells which were used for immunization and to one colon carcinoma, HT29. This monoclonal antibody is clearly reacting with an
TABLE 2 IMMUNOGLOBULINSUBCLASSOFHYBRIDOMAANTIBODIES Hybridoma
169D4 (cpm) 172DS (cpm) 179E3 (cpm)
40,341 7,568 6,708
666 5284 4678
433 685 752
556 708 1081
847 798 1111
602 669 537
IgM 31,961 416 1,253
Note. P3 cells (2 x lOV50 ~1) were incubated 30 min at 37°C with hybridoma culture supernatant and incubated with class-specific rabbit anti-mouse immunoglobulin (1:50) and then incubated with lZSI-protein A.
JOURNAL OF SURGICAL RESEARCH: VOL. 30, NO. 4, APRIL 1981
We have described the properties of three monoclonal antibodies, which showed clearly different reactivity patterns against cultured tumor cell lines, each specific for an antigenie determinant expressed on human lung carcinoma cells. However, in previous monoclonal antibody studies with human tumors particularly in the melanoma system, many antibodies were found to react against transplantation alloantigens, including HLA-A, -B, -C or -DR antigens. Recently, it was reported that cultured human tumor cells express DR antigens, which are usually expressed strongly on certain immune cells like B-lymphoid cells [15, 161. For this rea-
FIG. 2, Target cell suspension assay using lz51protein A assay of hybridoma 169D4 antibody. The hybridoma antibody was tested for binding to various tumor and nontumor cells.
antigenic determinant with a very narrow specificity. Figure 3 shows that the antibody from hybridoma 172D5 bound to three out of six lung carcinomas, two colon carcinomas and three breast carcinomas, but bound at low or undetectable levels to melanomas, sarcoma, embryonic cells, B-lymphoblastoid lines, and peripheral blood lymphocytes. This antibody clearly reacts with a tumorspecific antigen on many of the tested carcinomas. The antibody from hybridoma 179E3 shows the broadest cross-reactivity and binds to all the tested tumors and embryonic cell lines except for sarcomas, but did not react with lymphoblastoid lines and PBL. These antibodies need to be tested against a larger panel of other tumors and nontumor cells from biopsy specimens before a reliable statement about actual tumor specificity can be determined.
FIG. 3. Target cell suspension assay using “‘Iprotein A assay of hybridoma 172D5 antibody. The hybridoma antibody was tested for binding to various tumor and nontumor cells.
KASAI ET AL.: MONOCLONAL
erentially expressed on the cultured tumor cell lines. The antibody from hybridoma 172D5 recLung CO PI P3 ognizes an antigenic determinant which is P30 expressed on 8 out of 11 carcinomas tested P4 P6 and seems to recognize a carcinoma-associP7 ated antigen. However, the antigen is not 157 Eireost CO carcinoembryonic antigen (CEA) or cu-feto231 protein (AFP) because the P3 tumor cell 734 used for immunization is a squamous cell tin9 Colon co carcinoma which does not produce CEA or Don AFP in the spent culture medium (unpubMelonomo Ml0 lished data). MM On the other hand, the antibody from Ml5 M20 hybridoma 179E3 showed a broad crossreactivity with all the tumor and embryonic sorcomo s1 s3 cell lines tested except for sarcomas. The antigenic determinant detected by this WHE hb,p monoclonal antibody may be an oncofetal Lymphoblosl PL3 antigen. L14 L20 However, these monoclonal antibodies SLI must be tested against a larger panel of tumors and nontumors, and tested for crossPBL JK SG reactivity with specific antigens on biopsy NB tumor and normal tissues before a reliable BP QK statement about in viva tumor specificity OK can be made. We feel that this approach using paired FIG. 4. Target cell suspension assay using lz51protein A assay of hybridoma 179E3 antibody. The tumor and autologous B-cell lines to screen hybridoma antibody was tested for binding to various hybridoma monoclonal antibodies will eventumor and nontumor cells. tually prove to be a very useful method for defining unique cell surface components son, we compared hybridoma antibody on human tumor cells. binding to a human squamous cell carcinoma and an autologous HLA identical ACKNOWLEDGMENTS B-lymphoblastoid cell line as target cells We thank G. Worth, F. Parker, and C. Kosaka for using indirect protein A binding assay. By this screening assay, several antibodies excellent technical assistance, and B. Fink for preparation of the manuscript. from hybridomas were found to bind over fivefold more strongly to P3 tumor cells than to autologous PL3 lymphoblasts. These REFERENCES hybridomas were selected and cloned in 1. Accolla, R. S., Carrel, S., and Mach, J. P. Monoorder to exclude antibodies cross-reactive clonal antibodies specific for carcinoembryonic with HLA antigens. The monoclonal antiantigen and produced by two hybrid cell lines. Proc. Natl. Acad. Sci. USA. 77: 563, 1980. bodies shown in Figs. 2-4 displayed very 2. Brown, J. P., Tamerius, J. D., and Hellstrom, I. low or undetectable binding to autologous Indirect ‘251-labeled protein A assay for monolymphoblastoid cells, so the reactivity of clonal antibodies to cell surface antigens. J. Zmeach hybridoma antibody appears to be munol. Methods 31: 201, 1979. directed to an antigenic determinant pref3. Brown, J. P., Wright, P. W., Hart, C. E., Wood-
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