Immunol. Cell Bioi. (1990)68. 263-268
Monoclonal antibodies to carcino-embryonic antigen Jiti-Ghee Teh and Ian F. C. McKenzie Research Centre for Cancer and Transplantation. Department of Pathology, University of Melbourne, Vic. 3052. Australia (Submitted
19 June 1990. Accepted for publication
16 July
1990.)
Summary Several approaches were made to produce new monoclonal antibodies (MoAb) to human colonic tumours using different immunogens, such as colon cell lines, and tissue and serum from patients with colon cancer. Six MoAb were produced but al! were found lo be reactive with carcino-embryonic antigen {CEA). Through tissue reactivity and competitive inhibition studies it was found that four epitopes could be detected: CEA-I detected by MoAb I-l and O-l; CEA-2 detected by MoAb i7C4; CEA-3 detected by MoAb LK-4 and XPX-13 and CEA-4 detected by MoAb JGT-13. CEA-2 was also found on granulocytes and probably detects normal cross-reacting anligen (NCA). In formalin-fixed tissues the CEA-specific epitopes were absent from normal adult tissues, but this was an artefact of ihc fixation procedure and fresh, non-fixed colonic tissues were clearly reactive. All six MoAb reacted with > 70% of colonic tumours when tested by the immunoperoxidase teehnique and gave different patterns of reactivity reflecting the differential expression of lhe CEA 1-4 epitopes. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to CEA polyclonal antibodies, that is the MoAb gave no obvious advantage. Thus efforts to produce new reagents to colon cancer yielded only anti-CEA monoclonal reagents. Clearly, other new approaches are required to produce better diagnostic reagents for this common disease.
INTRODUCTION Colon cancer is a common disease affecting approximately I in 20 Australians in their lifetime. In addition, early diagnosis is accompanied by a favourable prognosis, whereas when the disease has spread beyond the eolon. the prognosis is poor and there is virtually no effective treatment available. Unfortunately, there are no useful early diagnostic tests available — blood tests for carcino-embryonic antigen (CEA) are insensitive in early disease, and faecal tests for blood are too non-specific. We have therefore developed a programme seeking new reagents to detect colon eancer, and have modified the immunization programme to include different sources of tissue. However, as will be shown. anti-CEA antibodies invariably appear. In the belief that monoclonal anti-CEA antibodies might be more specific tban polyelonal antibodCorrespondence: J-G. Teh, Research Centre for Cancer and Transplantation. Department of Pathology, University of Melbourne, Vic. 3052, Australia. Abbreviations used in this paper: MoAb. monoclonal antibodies; CEA. carcino-embryonic antigen; NCA, normal cross-reacting antigen; ELISA, enzyme-linked immunosorbent assay.
ies., the anti-CEA antibodies were characterized and compared with an existing anti-CEA test kit. It was clear that although these reagents appear to be highly speeifie on tissue sections for colon eancer. serum testing with monoclonal antibodies (MoAb) gave no advantage over the polyclonal antibodies. Other approaches are necessary to produce new diagnostic reagents for colon cancer.
MATERIALS AND METHODS Cell lines were grown as described (I); those to be tested by the immunoperoxidase technique were grown on coverslips. washed, air dried, fixed in 10% formalin and treated with 5%H;O; for20 min ai room temperature prior to use. Cell lines used for enzymelinked immunosorbent assays (ELISA) were grown on flat bottomed tissue culture plates and fixed with 0-25%glutaraldehyde before testing. Fifty microlitres of MoAb supernatant was added for 30 min and after washing, 50|iL of a 1:200 dilution of sheep antimouse antibody conjugated to horseradish peroxidase {Amersham. UK) was added for 30 min and washed. ELISA were developed with 50/iL of 0 03% 2.2'azino-di-(3-ethylbenzthiazoline sulfate) {Amersham. UK) and the optical density at 405 nm was measured with a multisean automated plate reader {Flow Laboratories. Australia).
J-G. TEH AND [. F. C. MCKENZIE
264
MoAb I-l and 0-1 were produced as described (2). MoAb 17C4 was produced by immunizing a {CBAXBALB/C)FI mouse wilh the colonic cell line COLO 397 on days 0 and 5 and with cell suspensions of a primary colonic tumour on days 21 and 29 before fusion on day 32. MoAb JGT-13. LK-4 and XPX-13 were produced by immunizing a (CBAXBALB/C)FI mouse with a pool of eight cell lines, one fresh colonic tumour and serum samples from four patients with colonic tumours on days [. 5. 8. 32. 35. 51 and 54 followed by fusion on day 57. Fusion was performed with murine myeloma P3-NS-l-Ag4-l (3). Hybrids were screened by immunoperoxidase testing on formalin-fixed sections of colonic carcinoma and cloned by limiting dilution. MoAb 17C4 wasof IgG2b isotype and the others were of lgGi isotype by Ouchterlony assay. MoAb 30-6 (IgG^a) was used as a negative control MoAb: it was raised against coiorectal carcinoma and was reactive with fresh tissues but not formalinfixed tissues or CEA; (1). Immunoperoxidase staining was performed on formalin-fixed tissues (3) using horseradish peroxidase directly eonjugated lo rabbit anti-mouse antibodies (Dako, Uppsala. Sweden). Flow cytometry was performed on peripheral blood cells using an Ortho 50 H Cytofluorograph and fluorescein conjugated sheep anti-mouse antibodies (Silenus. Australia): by flow cytometry. granulocytes. monoeytes and lymphocytes could be separately identified. Rosetting assays on cell lines were performed as previously described. The reactivity ofthe MoAb with CEA (both purified and in serum) was assayed as described (2). Briefly. radiolabeiled rabbit anti-CEA antibody was incubated with CEA or serum samples for 3 h. MoAb coated to beads were added for I h, washed and counted for radioactivity. Similarly, the reactivity of MoAb to different epitopes of CEA was examined by adding 1 mg of purified competitive MoAb for I h at room temperature to the radiolabeiled rabbit anti-CEA and CEA (100 /iL of 100 ng/mL) for 3 h followed by the addition of MoAb coated to beads. Normal human sera were obtained from the Melbourne blood bank and sera from patients with colonie eancer were obtained from the National Institute of Health, USA.
RESULTS Anti-CEA antibodies With the aim of producing new MoAb to coiorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonie tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb 1-1 and O-l were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens (2); when examined
E10 000
60
120
ngCEA/mL
Fig. 1. Graph of the ct/min bound of radioiabelled rabbit anti-CEA previously incubated with CEA. precipitated with beads eoupled with MoAb 1-1 (-H-). O-l (-•-). 17C4 (-•-), LK-4 (-0-), XPX-12 (-X-). JGT-13 i-o-). and 30-6 (-.4^), plotted against the concentration of CEA used in ng/mL.
in greater detail it was found that all these MoAb were reactive against the well characterized antigen CEA. MoAb LK-4. XPX-13. JGT-13. O-l. I-I and 17C4 were found to detect CEA as seen in Fig. 1 where they all precipitated increasing counts when increasing amounts of CEA were added {>8000 ct/min of radioiabelled rabbit-anti CEA was detected when incubated with 100 ng/mL of CEA). Negative control MoAb 30-6 precipitated
c
Z3000
u 520 000 n E
s
0
012600
/
10 000
* 10000
20 000 30000 17C4 CEA test
30 000 17C4 CEA lest
40 000
2200 1200
1400
1600
1800
2000
2200
2400
17C4 CEAtesl
40 000
Fig. 3. Graph of the ct/min bound of rabbit-antiCEA incubated with different scrum samples: (A) From five colonic cancer patients; (B) from four patients with benign colonic lesions; (C) from six patients with pancreatic cancer; (D) from six normal human sera; (E) the results of A-D pooled together. (A)-(E) all detected by MoAb 17C4 (.\-axis) compared with that obtained with a Pharmacia CEA-RIA 100 test kit (v-axis). Note that the scale from (D) is substantially different from the others. The statistical result of each graph was calculated by the computer program. Carcinoma of the colon sera (Q); benign lesions (-I-); carcinoma ofthe pancreas (A); NH sera (•).
267
MONOCLONAL ANTIBODIES TO CEA
epitope: CEA-3. Finally MoAb JGT-13 was able to block itself but I7C4.1-l. 5C1, XPX-I3 and LK-4 could not. defining another epitope designated CEA-4 (Fig. 2F). CEA serum tests: Monoclonal versus polyclonal antibody It was apparent that the different immunization procedures had yielded only anti-CEA antibodies and that these fell into four groups, based on differences in tumour staining and reactivity, which defined four different epitopes. A crucial question to be answered was whether any of these epitopes. detected by MoAb gave rise to a more specific and informative serum test for CEA than the polyclonal based antibody tests currently used. For example, we wished to determine whether detection of CEA-2 (for example) was more informative than detecting CEA-1, -3 or -4 or other epitopes (presumably detected by the polyclonal antibodies). On testing selected serum samples with known CEA levels determined by a Pharmacia CEA-RIA 100 test kit (Fig. 3), it was found that MoAb 17C4 detected serum samples (>3500 ct/min) from three of five colonic cancer patients (Fig. 3A), two of four patients with benign colonic lesions (Fig. 3B), four of six patients with pancreatic cancer (Fig. 3C) and none of six normal human sera (Fig. 3D). All samples which were positive with MoAb I7C4 were also positive with the Pharmacia test for CEA (Fig. 3E) and with MoAb 1-1 (2). Hence, even though the MoAb may detect some colonic tumours, it also detects serum samples of patients with pancreatic tumours and benign colonic lesions and is not specific for colonic cancer. This was also found with MoAb I-l (2) where scrum samples from patients with pancreatic tumours or benign colonic lesions were also positive. MoAb JGT-13, LK-4 and XPX-13 were also tested on some ofthe cotonic serum samples and it was found that high values were obtained where high CEA levels were detected by the other MoAb and by the Pharmacia test, and low levels were detected (
Monoclonal antibodies to carcino-embryonic antigen Jiti-Ghee Teh and Ian F. C. McKenzie Research Centre for Cancer and Transplantation. Department of Pathology, University of Melbourne, Vic. 3052. Australia (Submitted
19 June 1990. Accepted for publication
16 July
1990.)
Summary Several approaches were made to produce new monoclonal antibodies (MoAb) to human colonic tumours using different immunogens, such as colon cell lines, and tissue and serum from patients with colon cancer. Six MoAb were produced but al! were found lo be reactive with carcino-embryonic antigen {CEA). Through tissue reactivity and competitive inhibition studies it was found that four epitopes could be detected: CEA-I detected by MoAb I-l and O-l; CEA-2 detected by MoAb i7C4; CEA-3 detected by MoAb LK-4 and XPX-13 and CEA-4 detected by MoAb JGT-13. CEA-2 was also found on granulocytes and probably detects normal cross-reacting anligen (NCA). In formalin-fixed tissues the CEA-specific epitopes were absent from normal adult tissues, but this was an artefact of ihc fixation procedure and fresh, non-fixed colonic tissues were clearly reactive. All six MoAb reacted with > 70% of colonic tumours when tested by the immunoperoxidase teehnique and gave different patterns of reactivity reflecting the differential expression of lhe CEA 1-4 epitopes. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to CEA polyclonal antibodies, that is the MoAb gave no obvious advantage. Thus efforts to produce new reagents to colon cancer yielded only anti-CEA monoclonal reagents. Clearly, other new approaches are required to produce better diagnostic reagents for this common disease.
INTRODUCTION Colon cancer is a common disease affecting approximately I in 20 Australians in their lifetime. In addition, early diagnosis is accompanied by a favourable prognosis, whereas when the disease has spread beyond the eolon. the prognosis is poor and there is virtually no effective treatment available. Unfortunately, there are no useful early diagnostic tests available — blood tests for carcino-embryonic antigen (CEA) are insensitive in early disease, and faecal tests for blood are too non-specific. We have therefore developed a programme seeking new reagents to detect colon eancer, and have modified the immunization programme to include different sources of tissue. However, as will be shown. anti-CEA antibodies invariably appear. In the belief that monoclonal anti-CEA antibodies might be more specific tban polyelonal antibodCorrespondence: J-G. Teh, Research Centre for Cancer and Transplantation. Department of Pathology, University of Melbourne, Vic. 3052, Australia. Abbreviations used in this paper: MoAb. monoclonal antibodies; CEA. carcino-embryonic antigen; NCA, normal cross-reacting antigen; ELISA, enzyme-linked immunosorbent assay.
ies., the anti-CEA antibodies were characterized and compared with an existing anti-CEA test kit. It was clear that although these reagents appear to be highly speeifie on tissue sections for colon eancer. serum testing with monoclonal antibodies (MoAb) gave no advantage over the polyclonal antibodies. Other approaches are necessary to produce new diagnostic reagents for colon cancer.
MATERIALS AND METHODS Cell lines were grown as described (I); those to be tested by the immunoperoxidase technique were grown on coverslips. washed, air dried, fixed in 10% formalin and treated with 5%H;O; for20 min ai room temperature prior to use. Cell lines used for enzymelinked immunosorbent assays (ELISA) were grown on flat bottomed tissue culture plates and fixed with 0-25%glutaraldehyde before testing. Fifty microlitres of MoAb supernatant was added for 30 min and after washing, 50|iL of a 1:200 dilution of sheep antimouse antibody conjugated to horseradish peroxidase {Amersham. UK) was added for 30 min and washed. ELISA were developed with 50/iL of 0 03% 2.2'azino-di-(3-ethylbenzthiazoline sulfate) {Amersham. UK) and the optical density at 405 nm was measured with a multisean automated plate reader {Flow Laboratories. Australia).
J-G. TEH AND [. F. C. MCKENZIE
264
MoAb I-l and 0-1 were produced as described (2). MoAb 17C4 was produced by immunizing a {CBAXBALB/C)FI mouse wilh the colonic cell line COLO 397 on days 0 and 5 and with cell suspensions of a primary colonic tumour on days 21 and 29 before fusion on day 32. MoAb JGT-13. LK-4 and XPX-13 were produced by immunizing a (CBAXBALB/C)FI mouse with a pool of eight cell lines, one fresh colonic tumour and serum samples from four patients with colonic tumours on days [. 5. 8. 32. 35. 51 and 54 followed by fusion on day 57. Fusion was performed with murine myeloma P3-NS-l-Ag4-l (3). Hybrids were screened by immunoperoxidase testing on formalin-fixed sections of colonic carcinoma and cloned by limiting dilution. MoAb 17C4 wasof IgG2b isotype and the others were of lgGi isotype by Ouchterlony assay. MoAb 30-6 (IgG^a) was used as a negative control MoAb: it was raised against coiorectal carcinoma and was reactive with fresh tissues but not formalinfixed tissues or CEA; (1). Immunoperoxidase staining was performed on formalin-fixed tissues (3) using horseradish peroxidase directly eonjugated lo rabbit anti-mouse antibodies (Dako, Uppsala. Sweden). Flow cytometry was performed on peripheral blood cells using an Ortho 50 H Cytofluorograph and fluorescein conjugated sheep anti-mouse antibodies (Silenus. Australia): by flow cytometry. granulocytes. monoeytes and lymphocytes could be separately identified. Rosetting assays on cell lines were performed as previously described. The reactivity ofthe MoAb with CEA (both purified and in serum) was assayed as described (2). Briefly. radiolabeiled rabbit anti-CEA antibody was incubated with CEA or serum samples for 3 h. MoAb coated to beads were added for I h, washed and counted for radioactivity. Similarly, the reactivity of MoAb to different epitopes of CEA was examined by adding 1 mg of purified competitive MoAb for I h at room temperature to the radiolabeiled rabbit anti-CEA and CEA (100 /iL of 100 ng/mL) for 3 h followed by the addition of MoAb coated to beads. Normal human sera were obtained from the Melbourne blood bank and sera from patients with colonie eancer were obtained from the National Institute of Health, USA.
RESULTS Anti-CEA antibodies With the aim of producing new MoAb to coiorectal carcinoma, immunization with cell suspensions of a fresh colonic tumour was performed and MoAb 17C4 was obtained. To produce other MoAb to colon cancer, an immunization protocol using fresh tumour, colonic cell lines and sera from patients with colonie tumours was employed and resulted in MoAb JGT-13, LK-4 and XPX-13. MoAb 1-1 and O-l were raised against sera from patients with colon cancer to produce MoAb directed against circulating tumour associated antigens (2); when examined
E10 000
60
120
ngCEA/mL
Fig. 1. Graph of the ct/min bound of radioiabelled rabbit anti-CEA previously incubated with CEA. precipitated with beads eoupled with MoAb 1-1 (-H-). O-l (-•-). 17C4 (-•-), LK-4 (-0-), XPX-12 (-X-). JGT-13 i-o-). and 30-6 (-.4^), plotted against the concentration of CEA used in ng/mL.
in greater detail it was found that all these MoAb were reactive against the well characterized antigen CEA. MoAb LK-4. XPX-13. JGT-13. O-l. I-I and 17C4 were found to detect CEA as seen in Fig. 1 where they all precipitated increasing counts when increasing amounts of CEA were added {>8000 ct/min of radioiabelled rabbit-anti CEA was detected when incubated with 100 ng/mL of CEA). Negative control MoAb 30-6 precipitated
c
Z3000
u 520 000 n E
s
0
012600
/
10 000
* 10000
20 000 30000 17C4 CEA test
30 000 17C4 CEA lest
40 000
2200 1200
1400
1600
1800
2000
2200
2400
17C4 CEAtesl
40 000
Fig. 3. Graph of the ct/min bound of rabbit-antiCEA incubated with different scrum samples: (A) From five colonic cancer patients; (B) from four patients with benign colonic lesions; (C) from six patients with pancreatic cancer; (D) from six normal human sera; (E) the results of A-D pooled together. (A)-(E) all detected by MoAb 17C4 (.\-axis) compared with that obtained with a Pharmacia CEA-RIA 100 test kit (v-axis). Note that the scale from (D) is substantially different from the others. The statistical result of each graph was calculated by the computer program. Carcinoma of the colon sera (Q); benign lesions (-I-); carcinoma ofthe pancreas (A); NH sera (•).
267
MONOCLONAL ANTIBODIES TO CEA
epitope: CEA-3. Finally MoAb JGT-13 was able to block itself but I7C4.1-l. 5C1, XPX-I3 and LK-4 could not. defining another epitope designated CEA-4 (Fig. 2F). CEA serum tests: Monoclonal versus polyclonal antibody It was apparent that the different immunization procedures had yielded only anti-CEA antibodies and that these fell into four groups, based on differences in tumour staining and reactivity, which defined four different epitopes. A crucial question to be answered was whether any of these epitopes. detected by MoAb gave rise to a more specific and informative serum test for CEA than the polyclonal based antibody tests currently used. For example, we wished to determine whether detection of CEA-2 (for example) was more informative than detecting CEA-1, -3 or -4 or other epitopes (presumably detected by the polyclonal antibodies). On testing selected serum samples with known CEA levels determined by a Pharmacia CEA-RIA 100 test kit (Fig. 3), it was found that MoAb 17C4 detected serum samples (>3500 ct/min) from three of five colonic cancer patients (Fig. 3A), two of four patients with benign colonic lesions (Fig. 3B), four of six patients with pancreatic cancer (Fig. 3C) and none of six normal human sera (Fig. 3D). All samples which were positive with MoAb I7C4 were also positive with the Pharmacia test for CEA (Fig. 3E) and with MoAb 1-1 (2). Hence, even though the MoAb may detect some colonic tumours, it also detects serum samples of patients with pancreatic tumours and benign colonic lesions and is not specific for colonic cancer. This was also found with MoAb I-l (2) where scrum samples from patients with pancreatic tumours or benign colonic lesions were also positive. MoAb JGT-13, LK-4 and XPX-13 were also tested on some ofthe cotonic serum samples and it was found that high values were obtained where high CEA levels were detected by the other MoAb and by the Pharmacia test, and low levels were detected (
