GYNECOLOGIC
ONCOLOGY
37, 24-28 (1990)
Ovarian Cancer-Associated Antibodies Recovered from Ascites: Their Use for the Isolation of Ovarian Cancer-Associated Antigen to Produce Monoclonal Antibodies FRANCESCA
R. GIANCOTTI, BRENT H. DORSETT, HENIAN QIAN, WILLIAM J. CRONIN, HUGH R. K. BARBER, AND HARRY L. IOACHIM
Department of Pathology, Lenox Hill Hospital and Columbia University College of Physicians and Surgeons, New York, New York 10021, and Department of Obstetrics and Gynecology, Lenox Hill Hospital and People’s Hospital, Beijing, People’s Republic of China
Received March 3, 1988 Immune complexes (ICs) were recovered from the ascites of a patient with stage IV endometrioid ovarian cancer by sequential precipitation with 33% saturated ammonium sulfate and 2.5% polyethylene glycol 6000 (PEG 6000), followed by affinity chromatography on protein A-Sepharose CL4B. The IgG-containing ICs were dissociated using 8 M urea, separated by ion-exchange chromatography on Sephadex QAEJO, and subsequently analyzed for purity by immunoelectrophoresis (IEP) and radial immunodlffusion (RID). Recovered antibody was tested for reactivity by immunohistologlc techniques against paraffin-embedded tumor tissue and acetone-fixed cell suspensions of epithelial tumors. The antibody which demonstrated ovarian cancer-associated activity was absorbed with antigen extracts of breast, colon, and lung cancers as well as keratin to reduce cross-reactivity. The absorbed endometrioid ovarian cancer-associated antibody (CXXAb) was used to produce an immunoadsorbent column for the recovery of tumor-associated antigens. A mouse monoclonal antibody designated FEN-1 was produced using this antigencontaining fraction, and preliminary screening has demonstrated ovarian tumor-associated reactivity. The use of autologous ICs as reagents for preparing tumor antigen-rich immunogens may provide a valuable tool in the search for tumor-associated antigens. 0 1990 Academic Press, Inc. INTRODUCTION
Peritoneal effusions in patients with advanced ovarian cancer may be rich sources of immune complexes (ICs) which, when recovered and dissociated, contain tumorassociated antibodies and antigens [l-8]. The presence of circulating immune complexes (CICs) has been demonstrated in a number of disease states including a variety of malignancies [9-l 11.In some instances, the level of CICs has been correlated with the severity of the disease as well as patient prognosis [ 12-141. Ascitic fluid
MATERIALS
Copyright 0 1990by Academic Press,Inc. All rights of reproductionin any form reserved.
AND METHODS
Recovery and dissociation of ICs from peritoneal effusions. Ascitic fluid was collected from a patient with
stage IV (FIG0 classification) endometrioid cancer of 24
0090-8258/90$1.50
is similar in composition to serum, and is in immediate contact with the tumor; therefore, it might be expected to contain substantial levels of ICs. In our laboratory, we have previously demonstrated that ovarian cancerassociated antibodies (OCAAbs) recovered from such ICs are tumor associated, and that the corresponding antigens are immunoreactive [7,8,15-171. We have developed new methods allowing the recovery of sizable quantities of IC-associated antibodies and antigens from malignant effusions in cancer patients. These antigens were successfully employed as immunogens to prepare heterologous lung tumor-associated antisera [ 181. The production of monoclonal antibodies with tumor specificity holds great potential for immunodiagnosis, nuclear imaging, and immunotherapy of malignant disease. The selection of suitable immunogens for preparing such antibodies is of critical importance. Immunization with crude tumor cell extracts containing unknown quantities of antigens may limit the success of these procedures. The finding that autologous ICs from malignant effusions contain substantial amounts of tumor-related antibodies and antigens makes it possible to utilize the patient’s own humoral immune response to produce antigen-rich immunogens for monoclonal antibody preparation. This paper reports on the methods employed to produce ovarian cancer-associated monoclonal antibodies using ICs present in the ascites of a patient with stage IV endometrioid cancer of the ovary.
OVARIAN
CANCER
ANTIBODIES/ANTIGENS
the ovary and processed for the recovery of associated antibodies and antigens [16]. Briefly, an IgG-containing IC-rich fraction was prepared by sequential precipitation with saturated ammonium sulfate (Sigma Chemical Co., St. Louis, MO), and polyethylene glyco16000 (PEG 6000, Fischer Scientific Comp., Pittsburgh, PA), followed by affinity chromatography on a protein A-Sepharose CL4B column (Pharmacia Fine Chemicals, Piscataway, NJ). Immune complexes were dissociated in cold 8 M urea, and the antibody and antigen fractions were recovered by ion-exchange chromatography as previously described [16]. The total protein was determined by the method of Bradford [19]. The amounts and purity of IgG recovered were assessed by radial immunodiffusion (RID) and immunoelectrophoresis (IEP) as previously described [18]. Immunojluorescence against fixed Ovarian Cancer cells. The pK1 antibody fraction (OCAAbs) was tested against acetone-fixed cell suspensions of an endometrioid ovarian cancer cell line (L-l) established in our lab [20], by immunofluorescence (IF) to determine the titer as previously described [ 181. Solid-phase immunoabsorption of recovered antibodies. Antigen extracts were prepared from solid tumors of an adenocarcinoma of the breast, an adenocarcinoma of the colon, and a squamous cell carcinoma of the lung, and from a commercially prepared keratin powder (US Biochemical Corp., Control No. 18946). The solid tumors were minced, suspended in 10 ml of ice-cold phosphatebuffered saline (PBS) containing 1% Triton X-100, and ultrasonically disrupted with a 100-W MSE tissue disintegrater. The sonicates were clarified by centrifugation at 100,OOOgfor 60 min at 4°C and the protein concentration of the supernatant was determined as previously described. These antigen extracts were covalently linked to cyanogen bromide-activated Sepharose beads to produce solid-phase immunoabsorbents [21]. Approximately 20 mg of OCAAb (pK1) was repeatedly passed through the immunoabsorbent gel bed, collected, and stored at 4°C. Evaluation of ovarian cancer-associated antibody by immunohistology. Following solid-phase immunoabsorption, recovered antibody was tested against a panel of Bouins’ fixed, paraRm-embedded malignant tumor tissue sections by IF [22] and by the biotin-avidin immunoperoxidase technique [23]. Briefly, the sections were deparaffinized and incubated with OCAAb for 60 min, followed by 45min incubations with biotinylated goat antihuman IgG and peroxidase-conjugated avidin-biotin complex (Vector Lab, Burlingame, CA). The sections were subsequently developed with 3-amino-9-ethylcarbazole (AEC) substrate reagent (Dakopatts, Santa Barbara, CA). All incubations were proceeded with three washes in PBS. Control tissue sections were run in par-
IN MONOCLONAL
ANTIBODY
PRODUCTION
25
allel without primary antibody to assess background reactivity. Recovery of ovarian cancer-associated antigen by affinity chromatography with OCAAb. Absorbed OCAAb was dialyzed against coupling buffer and combined with swollen and equilibrated cyanogen bromide-activated Sepharose CL-4B gel at a ratio of 5 pg of protein to 1 ~1 of gel. The gel slurry was mixed by end-to-end rotation overnight at 4°C. Residual reactive groups were blocked and 3 ml of crude pKI1 antigen fraction with a total protein concentration of 8.9 mg was passed through the column three times. Absorbed ovarian cancer-associated antigen (OCAA) was eluted with 0.1 M glycine-HCl buffer, pH 2.8, containing 1 M NaCI, neutralized, pooled, and dialyzed against PBS at 4°C. Production of monoclonal antibodies. Affinity-purified OCAA (250 pg/ml) was combined with an equal volume of Freund’s complete adjuvant (v/v) and injected SCinto Balb/c mice. Ten days after immunization, the mice were bled and the antisera tested for reactivity and specificity against acetone-fixed L-l cells and Bouins’ fixed, paraffin-embedded malignant tumors by IF. The mouse whose antiserum demonstrated the strongest reactivity against endometrioid carcinoma of the ovary was sacrificed, and the spleen aseptically removed for the fusion of antigen-primed splenocytes with immortal SP2/Ag14 myeloma cells in the presence of PEG 1500 to produce hybridomas [24]. The resultant hybrids were screened for the production of immunoglobulins reactive with the immunizing antigen. One particular hybrid was subsequently cloned and is being maintained as a tissue culture cell line designated Ml7 secreting a monoclonal antibody (Moab), FEN-l, demonstrating ovarian cancer-associated reactivity. RESULTS Dissociation of ZCs and Recovery of OCAAb The pK1 antibody fraction (OCAAb) contained 0.26 mg/ml IgG as determined by RID, and only IgG as revealed by IEP. When tested against acetone-fixed L-l cells by IF, the endpoint titer of antibody activity was found to be 1: 128. Staining in the cytoplasm appeared diffuse and granular. Immunofluorescence staining was not confined within one region of the cytoplasm such as the perinuclear space. Zmmunohistology of OCAAb (pKZ) The unabsorbed OCAAb fraction was found to react with all three common epithelial ovarian carcinomasserous, endometrioid, and mutinous-by IP and IF on partin-embedded Bouins’ fixed tissue. The antibody, however, was found to cross-react with normal gastric
26
GIANCOTTI
mucosa, squamous cell carcinoma of the lung, as well as adenocarcinoma of the breast and colon. Immunofluorescence staining of serous cystadenocarcinoma of the ovary showed reactivity confined to the tumor site. Staining was cytoplasmic and no reactivity occurred in the surrounding tissue. Immunohistology of Affinity-PuriJed
OCAAb
Following immunoabsorption, OCAAb was evaluated for specificity and was found to strongly react with epithelial ovarian malignancies, both “in situ” and metastatic. Staining occurred with papillary serous tumors, as well as endometrioid ovarian carcinoma, but not mucinous tumors. Fluorescent staining demonstrated a strong intracytoplasmic diffuse pattern within the ovarian tumor cells. Absorption with breast and lung carcinomas removed cross-reactivity with squamous cell carcinoma of the lung; however, the antibody still showed some weak reactivity against adenocarcinoma of the breast. When tested against an adenocarcinoma of the colon, no reactivity was observed (Table 1). Evaluation of Immunized Mouse Antiserum Immuno-Fluorescence on a Bouins’ fixed, paraffinembedded endometrioid ovarian cancer section revealed strong cytoplasmic fluorescence staining of the tumor cells. No reaction was seen in the stroma or surrounding tissue. The antiserum’s staining pattern paralleled that of absorbed OCAAb (Table 1). Preliminary Evaluation of Monoclonal Antibody A mouse monoclonal antibody produced with affinityisolated OCAA was designated FEN- 1, and preliminary immunohistologic observation demonstrates reactivity against endometrioid and serous ovarian carcinomas. This antibody does not react against mutinous tumors, nor does it cross-react with gastrointestinal neoplasms. TABLE 1 Immunohistochemical Reactivity of Unabsorbed and Absorbed Autologous Antibodies and Immunized Mouse Serum Antibodies OCAAb
OCAAb
Tissue type
Peak I
Absorbed
Breast cancer Lung cancer Colon cancer Ovarian cancer Endometrioid Papillary Serous Mutinous
+ + +
+I-
+I-
+++ + +
+++ + -
+++ + -
Immunized mouse serum
ET AL.
TABLE 2 Immunohistochemical Reactivity of MoAb FEN-l Tissue
Number positive
Number tested
Ovarian tumors Endometrioid Serous Mutinous Clear cell Brenner Undifferentiated Mixed tumors Nonepithelial Nongynecologic Tumors Normal tissue
26 20 4 0 0 0 0 2 0 1 1
75 25 8 6 3 2 2 3 26 30 20
No reactivity was seen against fetal tissue, placenta, and normal tissue except goblet cells of normal colonic mucosa. MoAb FEN-l did not react against lymphoid neoplasms, sarcomas, melanoma, or inflammatory and mesothelial cells (Table 2). The antibody reacted with 80% of the endometrioid ovarian carcinomas tested on fixed tissue. DISCUSSION
The presence of tumor-associated immunoglobulins in ascitic fluids of patients with ovarian cancer has been demonstrated [3-8, 25-291. These immunoglobulins are found in free form or bound to antigens in the form of ICs. The ICs are of multiple specificities and may be continuously shed from the surface of the tumor itself [30]. The presence of ICs in ascitic fluids of women with advanced cancer of the ovary is not unexpected since the fluid accumulates in close proximity to the solid tumor mass [31]. We, as well as other investigators, have shown that there is an autologous humoral immune response to the tumor in ovarian cancer [3-8,321. Immunoglobulins recovered from ICs of patients with ovarian cancer have demonstrated autologous tumor-associated activity but not against normal ovary [3]. Isolated IgG (OCAAb) recovered from the IC fraction, has demonstrated ovarian cancer-associated reactivity by immunohistologic techniques. Cross-reactive components were removed from OCAAb by absorption with extracts of solid tumor since numerous tumor markers are associated with ovarian cancer [33]. OCAAb was absorbed with adenocarcinoma of the colon to minimize CEArelated activity, which has been found to be associated with cancer of the ovary [34]. In fact, absorbed OCAAb and immunized mice antiserum were found not to react against colon cancer and mutinous tumors of the ovary. We were able to successfully demonstrate that absorption did not reduce immunoreactivity. Initial results
OVARIAN
CANCER ANTIBODIES/ANTIGENS
showed that autologous affinity-purified ovarian tumor antigens can be used as immunogens since mice immunized with such immunogens produced antiserum staining patterns similar to those of autologous IC-associated antibodies. Preliminary results have shown that MoAb FEN-l, like absorbed OCAAb and immunized mouse antiserum, demonstrates ovarian cancer-associated reactivity by immunohistology, particularly against endometrioid tumors. With the immunogen preparation described here, it is possible to use autologous antigen fractions recovered with the patient’s own reactive antibodies for the production of MoAbs demonstrating tumor-associated reactivity. This method for recovering an immunogen to produce MoAbs may provide a means to identify the possible existence of tumor-specific rather than tumor-associated antigens in human tumors, particularly ovarian cancer. ACKNOWLEDGMENTS This project was made possible due to the generous support of the Emil and Zerline Hahnloser-Richard BAK Fund, the Hugoton Foundation, and the Hess Family Foundation, Inc.
REFERENCES 1. Clarke, A. G., Vasey, D. P., Symonds, E. M., Faratian, B., McLaughlin, P. J., Price, M. R., and Baldwin, R. W. Levels of circulating immune complexes in patients with ovarian cancer, Brir. J.’ Obstet. Gynaecol. 89, 231 (1982). 2. Clayton, L. A., Gall, S. A., Dawson, J. R., and Creasman, W. T. Immune complexes in ovarian cancer, Gynecol. Oncol. 13, 203 (1982). 3. Dorsett, B. H., Ioachim, H., and Walker, S. Isolation of tumorspecific antibodies from effusions of ovarian carcinoma, Znr. J. Cancer 16, 779-786 (1975). 4. Hill, R., Daunter, B., Khoo, S. K., and Mackay, E. V. Isolation of turnout--associated immunoglobulins from ascitic fluid, &it. J. Cancer 38, 154-157 (1978). 5. Silbum, P. A., Khoo, S. K., Hill, R., Daunter, B., and Mackay, E. V. Demonstration of tumor-associated immunoglobulin-G isolated from immune complexes in ascitic fluid of ovarian cancer, Diagn. Immunol. 2, 30-35 (1984). 6. Qian, H., Feng, J., and Fu, T. The study of antibodies and antigens dissociated from immune complexes extracted from ovarian cancer ascitic fluid, Gynecol. Oncol. 20, 100-108 (1985). I. Cantarow, W. D., Cronin, W. J., Dorsett, B. H., et al. Characterization of ovarian cancer antigen and antibody from immune complexes, Fed. Proc. 39, 474 (1980). 8. Stolbach, L. L., Cantarow, W. D., Cronin, W. J., et al. Dissociation and characterization of ovarian cancer antigen (Ag) and antibody (Ab) from immune complexes, Proc. Amer. Assoc. Cuncer Res. 21, 239 (1980). 9. Theophilopoulos, A. N., and Dixon, F. J. Immune complexes associated with neoplasia, in Immunodiagnosis of cancer (R. B. Heberman and K. R. Mcintire, Eds.), Marcel Dekker, New York, Vol. 9, Part 2 (1979). 10. Rayner, A. A., Berkowitz, R., Steele, G., Schur, P. H., Rodrick,
IN MONOCLONAL
ANTIBODY
PRODUCTION
27
M. L., Goldstein, D. P., Harte, P. J., Wilson, R. E., Zamcheck, N., and Munroe, A. E. Circulating immune complex levels in patients with gestational trophoblastic neoplasia, .I. Natl. Cancer Inst. 69, 23-26 (1982). 11. Gilead, Z., Troy, F. A., and Sulitzeanu, D. Isolation and electrophoretic analysis of immune complexes from patients with breast cancer, Eur. J. Cancer Clin. Oncol. 17, 1165-l 176 (1981). 12. Dodd, J. K., Hicks, L. J., Tyler, J. P. P., Crandon, A. J., and Hudson, C. N. Circulating IgG-specific immune complexes as a potential tumor marker in gynecological malignancies, Gynecol. Oncol. 16, 232-239 (1983). 13. Mooney, N. A., Tounsend, P. A., Wiltshaw, I., Evans, D. G., Shanti, R. K., and Pot&on, T. A. An assessment of sequential measurements of immune complex levels in ovarian cancer, Gynecol. Oncol. 15, 207-213 (1983). 14. Dodd, J. K., Tyler, J. P. P., Crandon, A. J., Blumenthal, N. J., Fay, R. A., Baird, P. J., Hicks, L. J., and Hudson, C. N. The value of the monoclonal antibody (cancer antigen 125) in serial monitoring of ovarian cancer, Brit. J. Obstet. Gynecol. 92, 10541060 (1985). 15. Ioachim, H. L., Dorsett, B. H., Sabbath, M., Anderson, B., and Barber, H. R. K. Antigenic and morphologic properties of ovarian carcinoma, Gynecol. Oncol. 1, 130-142 (1973). 16. Barber, H. R. K., and Dorsett, B. H. Immunologic aspects of gynecologic cancer, Cancer 48, No. 2 (July 15, 1981). 17. Dorsett, B. H., and Ioachim, H. L. Common antigenic component in ovarian carcinomas: Demonstration by double diffusion and immunofluorescence techniques, Immunol. Commun. 2, 173-184 (1973). 18. Cronin, W. J., Dorsett, B. H., and Ioachim, H. L. Isolation of lung carcinoma-associated antibodies from immune complexes and production of heterologous antisera, Caner Res. 42, 292-300 (1982). 19. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72, 248-254 (1976). 20. Giancotti, F. R., Dorsett, B. H., Weaver, S. C., Ioachim, H. L., and Barber, H. R. K. Description and characterization of an endometrioid ovarian cancer cell line. Gynecol. Oncol. 35, 330-337 (1989). 21. Axen, R., and Porath, J. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides, Nature (London) 214, 1302-1304 (1967). 37 Dorsett, B. H., and Ioachim, H. L. A method for the use of immunofluorescence on paraffin-embedded tissue, Amer. J. Clin. Puthol. 69, 66-72 (1978). 23. Hsu, S. M., Raine, L., and Fanger, H. The use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem. 29, 577-580 (1981). 24. Kohler, G., and Milstein, C. Continuous cultures of fused cells secreting antibody of pre-defined specificity, Nature (London) 256, 495-497 (1975). 25. Lutz, P., and Dawson, J. R. Activity of antibodies recovered from immune complexes of ovarian cancer patients, Cancer Immunol. Immunother. 17, 180-189 (1984). 26. Gerber, M. A., Koffler, D., and Cohen, C. J. Circulating antibodies in patients with ovarian carcinoma, Gynecol. Oncol. 5, 228 (1977). 27. Witz, I. Tumor-bound immunoglobulins: In situ expressions of humoral immunity, Adv. Cnncer Res. 25, 95-148 (1977). 28. Silbum, P. A., Neil, J. C., Khoo, S. K., Daunter, B., Hill, R., Wm.
28
GIANCOTTI
Collins, R. J., and Mackay, E. V. Immune complexes in ovarian cancer: Association between IgM class complexes and antinuclear autoantibodies in ascitic fluid, Int. Arch. Allergy Appl. Immunol. 74, 63-66 (1984). 29. Whitehouse, J. M. A. Circulating antibodies in human malignant disease, &it. J. Cancer 28, 170 (1973). 30. Sulitzeanu, D., Bercovici, B., Gosky, Y., and Levij, I. S. Membrane-bound immunoglobulin cells in effusions from patients with malignant disease, Isr. .I. Med. Sci. 12, 1330-1331 (1976). 31. Silbum, P. A., Khoo, S. K., Daunter, B., Hill, R., Roberts, T. K., and Mackay, E. V. Types of immune complexes in the ascitic fluid
ET AL. of women with carcinoma of the ovary, Int. Arch. Allergy Appl. 71, 219-223 (1983). 32. Benson, M. D., Lurain, J. R., and Newton, M. Ovarian tumor antigens, J. Reprod. Immunol. 28, 17-23 (1983). 33. Yamashita, K., Aitio, M. L., and Dawson, J. R. Characterization of CEA-like activity in cyst fluids from ovarian cancer patients, Cancer Res. 39, 1760-1765 (1979). 34. Hill, R., Khoo, S. K., Daunter, B., Silbum, P. A., and Mackay, E. V. Immunoglobulins reactive to CEA and their relationship to the antigen in malignant ascitic fluid of ovarian carcioma, Int. J. Cancer 30, 587-592 (1982). Immunol.
ONCOLOGY
37, 24-28 (1990)
Ovarian Cancer-Associated Antibodies Recovered from Ascites: Their Use for the Isolation of Ovarian Cancer-Associated Antigen to Produce Monoclonal Antibodies FRANCESCA
R. GIANCOTTI, BRENT H. DORSETT, HENIAN QIAN, WILLIAM J. CRONIN, HUGH R. K. BARBER, AND HARRY L. IOACHIM
Department of Pathology, Lenox Hill Hospital and Columbia University College of Physicians and Surgeons, New York, New York 10021, and Department of Obstetrics and Gynecology, Lenox Hill Hospital and People’s Hospital, Beijing, People’s Republic of China
Received March 3, 1988 Immune complexes (ICs) were recovered from the ascites of a patient with stage IV endometrioid ovarian cancer by sequential precipitation with 33% saturated ammonium sulfate and 2.5% polyethylene glycol 6000 (PEG 6000), followed by affinity chromatography on protein A-Sepharose CL4B. The IgG-containing ICs were dissociated using 8 M urea, separated by ion-exchange chromatography on Sephadex QAEJO, and subsequently analyzed for purity by immunoelectrophoresis (IEP) and radial immunodlffusion (RID). Recovered antibody was tested for reactivity by immunohistologlc techniques against paraffin-embedded tumor tissue and acetone-fixed cell suspensions of epithelial tumors. The antibody which demonstrated ovarian cancer-associated activity was absorbed with antigen extracts of breast, colon, and lung cancers as well as keratin to reduce cross-reactivity. The absorbed endometrioid ovarian cancer-associated antibody (CXXAb) was used to produce an immunoadsorbent column for the recovery of tumor-associated antigens. A mouse monoclonal antibody designated FEN-1 was produced using this antigencontaining fraction, and preliminary screening has demonstrated ovarian tumor-associated reactivity. The use of autologous ICs as reagents for preparing tumor antigen-rich immunogens may provide a valuable tool in the search for tumor-associated antigens. 0 1990 Academic Press, Inc. INTRODUCTION
Peritoneal effusions in patients with advanced ovarian cancer may be rich sources of immune complexes (ICs) which, when recovered and dissociated, contain tumorassociated antibodies and antigens [l-8]. The presence of circulating immune complexes (CICs) has been demonstrated in a number of disease states including a variety of malignancies [9-l 11.In some instances, the level of CICs has been correlated with the severity of the disease as well as patient prognosis [ 12-141. Ascitic fluid
MATERIALS
Copyright 0 1990by Academic Press,Inc. All rights of reproductionin any form reserved.
AND METHODS
Recovery and dissociation of ICs from peritoneal effusions. Ascitic fluid was collected from a patient with
stage IV (FIG0 classification) endometrioid cancer of 24
0090-8258/90$1.50
is similar in composition to serum, and is in immediate contact with the tumor; therefore, it might be expected to contain substantial levels of ICs. In our laboratory, we have previously demonstrated that ovarian cancerassociated antibodies (OCAAbs) recovered from such ICs are tumor associated, and that the corresponding antigens are immunoreactive [7,8,15-171. We have developed new methods allowing the recovery of sizable quantities of IC-associated antibodies and antigens from malignant effusions in cancer patients. These antigens were successfully employed as immunogens to prepare heterologous lung tumor-associated antisera [ 181. The production of monoclonal antibodies with tumor specificity holds great potential for immunodiagnosis, nuclear imaging, and immunotherapy of malignant disease. The selection of suitable immunogens for preparing such antibodies is of critical importance. Immunization with crude tumor cell extracts containing unknown quantities of antigens may limit the success of these procedures. The finding that autologous ICs from malignant effusions contain substantial amounts of tumor-related antibodies and antigens makes it possible to utilize the patient’s own humoral immune response to produce antigen-rich immunogens for monoclonal antibody preparation. This paper reports on the methods employed to produce ovarian cancer-associated monoclonal antibodies using ICs present in the ascites of a patient with stage IV endometrioid cancer of the ovary.
OVARIAN
CANCER
ANTIBODIES/ANTIGENS
the ovary and processed for the recovery of associated antibodies and antigens [16]. Briefly, an IgG-containing IC-rich fraction was prepared by sequential precipitation with saturated ammonium sulfate (Sigma Chemical Co., St. Louis, MO), and polyethylene glyco16000 (PEG 6000, Fischer Scientific Comp., Pittsburgh, PA), followed by affinity chromatography on a protein A-Sepharose CL4B column (Pharmacia Fine Chemicals, Piscataway, NJ). Immune complexes were dissociated in cold 8 M urea, and the antibody and antigen fractions were recovered by ion-exchange chromatography as previously described [16]. The total protein was determined by the method of Bradford [19]. The amounts and purity of IgG recovered were assessed by radial immunodiffusion (RID) and immunoelectrophoresis (IEP) as previously described [18]. Immunojluorescence against fixed Ovarian Cancer cells. The pK1 antibody fraction (OCAAbs) was tested against acetone-fixed cell suspensions of an endometrioid ovarian cancer cell line (L-l) established in our lab [20], by immunofluorescence (IF) to determine the titer as previously described [ 181. Solid-phase immunoabsorption of recovered antibodies. Antigen extracts were prepared from solid tumors of an adenocarcinoma of the breast, an adenocarcinoma of the colon, and a squamous cell carcinoma of the lung, and from a commercially prepared keratin powder (US Biochemical Corp., Control No. 18946). The solid tumors were minced, suspended in 10 ml of ice-cold phosphatebuffered saline (PBS) containing 1% Triton X-100, and ultrasonically disrupted with a 100-W MSE tissue disintegrater. The sonicates were clarified by centrifugation at 100,OOOgfor 60 min at 4°C and the protein concentration of the supernatant was determined as previously described. These antigen extracts were covalently linked to cyanogen bromide-activated Sepharose beads to produce solid-phase immunoabsorbents [21]. Approximately 20 mg of OCAAb (pK1) was repeatedly passed through the immunoabsorbent gel bed, collected, and stored at 4°C. Evaluation of ovarian cancer-associated antibody by immunohistology. Following solid-phase immunoabsorption, recovered antibody was tested against a panel of Bouins’ fixed, paraRm-embedded malignant tumor tissue sections by IF [22] and by the biotin-avidin immunoperoxidase technique [23]. Briefly, the sections were deparaffinized and incubated with OCAAb for 60 min, followed by 45min incubations with biotinylated goat antihuman IgG and peroxidase-conjugated avidin-biotin complex (Vector Lab, Burlingame, CA). The sections were subsequently developed with 3-amino-9-ethylcarbazole (AEC) substrate reagent (Dakopatts, Santa Barbara, CA). All incubations were proceeded with three washes in PBS. Control tissue sections were run in par-
IN MONOCLONAL
ANTIBODY
PRODUCTION
25
allel without primary antibody to assess background reactivity. Recovery of ovarian cancer-associated antigen by affinity chromatography with OCAAb. Absorbed OCAAb was dialyzed against coupling buffer and combined with swollen and equilibrated cyanogen bromide-activated Sepharose CL-4B gel at a ratio of 5 pg of protein to 1 ~1 of gel. The gel slurry was mixed by end-to-end rotation overnight at 4°C. Residual reactive groups were blocked and 3 ml of crude pKI1 antigen fraction with a total protein concentration of 8.9 mg was passed through the column three times. Absorbed ovarian cancer-associated antigen (OCAA) was eluted with 0.1 M glycine-HCl buffer, pH 2.8, containing 1 M NaCI, neutralized, pooled, and dialyzed against PBS at 4°C. Production of monoclonal antibodies. Affinity-purified OCAA (250 pg/ml) was combined with an equal volume of Freund’s complete adjuvant (v/v) and injected SCinto Balb/c mice. Ten days after immunization, the mice were bled and the antisera tested for reactivity and specificity against acetone-fixed L-l cells and Bouins’ fixed, paraffin-embedded malignant tumors by IF. The mouse whose antiserum demonstrated the strongest reactivity against endometrioid carcinoma of the ovary was sacrificed, and the spleen aseptically removed for the fusion of antigen-primed splenocytes with immortal SP2/Ag14 myeloma cells in the presence of PEG 1500 to produce hybridomas [24]. The resultant hybrids were screened for the production of immunoglobulins reactive with the immunizing antigen. One particular hybrid was subsequently cloned and is being maintained as a tissue culture cell line designated Ml7 secreting a monoclonal antibody (Moab), FEN-l, demonstrating ovarian cancer-associated reactivity. RESULTS Dissociation of ZCs and Recovery of OCAAb The pK1 antibody fraction (OCAAb) contained 0.26 mg/ml IgG as determined by RID, and only IgG as revealed by IEP. When tested against acetone-fixed L-l cells by IF, the endpoint titer of antibody activity was found to be 1: 128. Staining in the cytoplasm appeared diffuse and granular. Immunofluorescence staining was not confined within one region of the cytoplasm such as the perinuclear space. Zmmunohistology of OCAAb (pKZ) The unabsorbed OCAAb fraction was found to react with all three common epithelial ovarian carcinomasserous, endometrioid, and mutinous-by IP and IF on partin-embedded Bouins’ fixed tissue. The antibody, however, was found to cross-react with normal gastric
26
GIANCOTTI
mucosa, squamous cell carcinoma of the lung, as well as adenocarcinoma of the breast and colon. Immunofluorescence staining of serous cystadenocarcinoma of the ovary showed reactivity confined to the tumor site. Staining was cytoplasmic and no reactivity occurred in the surrounding tissue. Immunohistology of Affinity-PuriJed
OCAAb
Following immunoabsorption, OCAAb was evaluated for specificity and was found to strongly react with epithelial ovarian malignancies, both “in situ” and metastatic. Staining occurred with papillary serous tumors, as well as endometrioid ovarian carcinoma, but not mucinous tumors. Fluorescent staining demonstrated a strong intracytoplasmic diffuse pattern within the ovarian tumor cells. Absorption with breast and lung carcinomas removed cross-reactivity with squamous cell carcinoma of the lung; however, the antibody still showed some weak reactivity against adenocarcinoma of the breast. When tested against an adenocarcinoma of the colon, no reactivity was observed (Table 1). Evaluation of Immunized Mouse Antiserum Immuno-Fluorescence on a Bouins’ fixed, paraffinembedded endometrioid ovarian cancer section revealed strong cytoplasmic fluorescence staining of the tumor cells. No reaction was seen in the stroma or surrounding tissue. The antiserum’s staining pattern paralleled that of absorbed OCAAb (Table 1). Preliminary Evaluation of Monoclonal Antibody A mouse monoclonal antibody produced with affinityisolated OCAA was designated FEN- 1, and preliminary immunohistologic observation demonstrates reactivity against endometrioid and serous ovarian carcinomas. This antibody does not react against mutinous tumors, nor does it cross-react with gastrointestinal neoplasms. TABLE 1 Immunohistochemical Reactivity of Unabsorbed and Absorbed Autologous Antibodies and Immunized Mouse Serum Antibodies OCAAb
OCAAb
Tissue type
Peak I
Absorbed
Breast cancer Lung cancer Colon cancer Ovarian cancer Endometrioid Papillary Serous Mutinous
+ + +
+I-
+I-
+++ + +
+++ + -
+++ + -
Immunized mouse serum
ET AL.
TABLE 2 Immunohistochemical Reactivity of MoAb FEN-l Tissue
Number positive
Number tested
Ovarian tumors Endometrioid Serous Mutinous Clear cell Brenner Undifferentiated Mixed tumors Nonepithelial Nongynecologic Tumors Normal tissue
26 20 4 0 0 0 0 2 0 1 1
75 25 8 6 3 2 2 3 26 30 20
No reactivity was seen against fetal tissue, placenta, and normal tissue except goblet cells of normal colonic mucosa. MoAb FEN-l did not react against lymphoid neoplasms, sarcomas, melanoma, or inflammatory and mesothelial cells (Table 2). The antibody reacted with 80% of the endometrioid ovarian carcinomas tested on fixed tissue. DISCUSSION
The presence of tumor-associated immunoglobulins in ascitic fluids of patients with ovarian cancer has been demonstrated [3-8, 25-291. These immunoglobulins are found in free form or bound to antigens in the form of ICs. The ICs are of multiple specificities and may be continuously shed from the surface of the tumor itself [30]. The presence of ICs in ascitic fluids of women with advanced cancer of the ovary is not unexpected since the fluid accumulates in close proximity to the solid tumor mass [31]. We, as well as other investigators, have shown that there is an autologous humoral immune response to the tumor in ovarian cancer [3-8,321. Immunoglobulins recovered from ICs of patients with ovarian cancer have demonstrated autologous tumor-associated activity but not against normal ovary [3]. Isolated IgG (OCAAb) recovered from the IC fraction, has demonstrated ovarian cancer-associated reactivity by immunohistologic techniques. Cross-reactive components were removed from OCAAb by absorption with extracts of solid tumor since numerous tumor markers are associated with ovarian cancer [33]. OCAAb was absorbed with adenocarcinoma of the colon to minimize CEArelated activity, which has been found to be associated with cancer of the ovary [34]. In fact, absorbed OCAAb and immunized mice antiserum were found not to react against colon cancer and mutinous tumors of the ovary. We were able to successfully demonstrate that absorption did not reduce immunoreactivity. Initial results
OVARIAN
CANCER ANTIBODIES/ANTIGENS
showed that autologous affinity-purified ovarian tumor antigens can be used as immunogens since mice immunized with such immunogens produced antiserum staining patterns similar to those of autologous IC-associated antibodies. Preliminary results have shown that MoAb FEN-l, like absorbed OCAAb and immunized mouse antiserum, demonstrates ovarian cancer-associated reactivity by immunohistology, particularly against endometrioid tumors. With the immunogen preparation described here, it is possible to use autologous antigen fractions recovered with the patient’s own reactive antibodies for the production of MoAbs demonstrating tumor-associated reactivity. This method for recovering an immunogen to produce MoAbs may provide a means to identify the possible existence of tumor-specific rather than tumor-associated antigens in human tumors, particularly ovarian cancer. ACKNOWLEDGMENTS This project was made possible due to the generous support of the Emil and Zerline Hahnloser-Richard BAK Fund, the Hugoton Foundation, and the Hess Family Foundation, Inc.
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