APMIS 98: 674-684, 1990
Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens PER BORUP-CHRISTENSEN', KARIN ERB', HENRIK DITZEL', BJARNE NIELSEN3, J 0 R G E N K. LARSEN4, SVEN-ERIK SVEHAG2and JENS CHRISTIAN JENSENIUS' 'Institute of Surgery/Biomedical Laboratory, 'Institute of Medical Microbiology and 31nstitute of Pathology, University of Odense, Denmark, and 4Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
Borup-Christensen, P., Erb, K., Ditzel, H., Nielsen, B., Larsen, J. K., Svehag, S.E. & Jensenius, J. C . Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens. APMIS 98: 674-684, 1990. Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B-lymphoblastoid cell lines, LICR-LON-HMy-2 (HMy-2) and WI-L2-729-HF2 (729-HF2), to generate hybridomas synthesizing antibodies reacting with tumor-associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been furhter analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9 165 and D42 13)showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (FI 1348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G 4 I46 to two components from colon cancer cells with M, of 59 K and 6 1 K, while B9 165 bound to a 43 K component and FI 1348 to several components with M, from 30 to 200K. D4213 showd no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens. Key words: Human monoclonal antibodies; colorectal cancer: immunohistochemical analysis. Karin Erb, Institute of Medical Microbiology, University of Odense, J. B. Winslerwsvej 19, 5000 Odense, Denmark.
Colorectal cancer is one of the most frequent cancers and has a poor prognosis. It is therefore important that clinically useful tumor markers are identified. There have been several reports on murine monoclonal antibodies against human gastrointestinal carcinoma-associated antigens (4, 16,19,23,3 I), some of which may prove useful for this purpose. Therapeutic trials have been conducted with murine antibodies ( 6 , 2 9 , 33,36), but difficulties may be encountered since the adminis-
Received November 1 1, 1989. Accepted January 29, 1990.
tered antibodies can elicit an immune response in the patient. This problem may be circumvented by the use of human monoclonal antibodies, although anti-allotype or anti-idiotype reactions might still complicate the picture. A further potential advantage of human monoclonals could be the occurrence of tumor-associated antigens which are more immunogenic in humans than in other species (2 1). Data in the literature suggest that patients make antibodies that react with their own tumors (9, 13, 32, 41). Furthermore, Schlorn et al. (34) demonstrated, and others (14, 35) have confirmed, that immortalization of B-lymphocytes from the regional draining lymph nodes of cancer patients
HUMAN MONOCLONAL ANTIBODIES TO EDORECTAL CANCER
may result in cell lines synthesizing antibodies against tumor antigens. We have earlier described initial results from the fusion of lymphocytes from regional lymph nodes of patients with colorectal cancer with the human B-lymphoma cell line LICR-LON-HMy-2 (5). In the present work we compare these results with the outcome of fusions with another human cell line WI-L2-729-HF2 (l), and report further characterization of 4 human monoclonal antibodies, all of the IgM class.
MATERIALS AND METHODS Human IgM. Human myeloma IgM containing camer protein was procured from Cappel Laboratories, Cochranville, P.A. (Cat. No. 13334). Normal human polyclonal IgM was purified from serum by affinity chromatography on Sepharose 4B-coupled anti-p chain antibody (HB-57, American Type Culture Collection, MD (ATCC)). Myeloma IgM was purified similarly from a patient with Waldenstrom's macroglobulinaemia. Fusion cell lines. The human B-lymphoblastoid cell line LICR-LON-HMy-2 (HMy-2)( 12) wasagift from Dr M. J. O'Hare, The Ludwig Institute, London. It produces IgG, (kappa). The human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2) was obtained from Techniclone International, California, U.S.A. This cell line secretes no immunoglobulin (1). Both cell lines were prior to fusion grown in RPMI1640 medium (GIBCO, Grand Island, N.Y.) supplemented with 10% fetal calf serum (FCS, GIBSO), 100 units penicillin and 100 pg streptomycin per ml. HMy-2 was grown with 4 mM 8-azaguanine (Sigma, St. Louis, Mo., U.S.A.) ( 12) and 729-HF2 with 0.1 mM 6-thioguanine (Sigma) (1). Human l.vmphocytes. Lymphocytes from the mesenteric lymph nodes draining the tumor region in patients with colorectal cancer were purified by centrifugation on Ficoll-Isopaque (Boehringer-Mannheim, Mannheim, FRG). Lymphocytes used for fusion with 729-HF2 were used without prior stimulation, while pokeweed mitogen (2.5 pg/ml) stimulated lymphocytes were used for fusion with HMy-2 (5). Fusion procedure. Fusion was performed according to the methodsdescribed by Kohler (24). The ratio between HMy-2 or 729-HF2 and lymphocytes ( 107-108)was 1:2, and the cells were plated at 2 x lo5cells in 200 pl per well of 96-well microplates (Costar, cat. no. 3596, Cambridge, Mas., U.S.A.). Lymphocytes fused with HMy-2 were seeded on plates with feeder cells (12). Feeder cells are not necessary when 729-HF2 is used (1). The cells fused with HMy-2 were maintained in HAT medium (2 x M hypoxanthine, 4 x 10.' M aminopterine, 3.2 x 1O-j M thymidine) for 6 weeks (12), while 729-HF2 fusion products were selected in HAT or
AH (5.8 x M azaserine, 2 x M hypoxanthine) medium for 2 weeks ( 1). AH was reported to be superior to HAT for selection of murine hybridomas(22). Further cultivation was in RPMI- 1640, I OYo FCS, supplemented with hypoxanthine when AH was used as selection medium. Growing hybrids appeared from 3 to 6 weeks after fusion with HMy-2 and 10 days to 4 weeks after fusion with 729-HF2. Cloning by limiting dilution was carried out with feeder cells when using HMy-2 and without feeder cells for 729-HF2. Immunoglobulin assays. Supernatants from wells with growing clones were assayed for human IgM and IgG by enzyme linked immunosorbent assay (ELISA). Immunoplates (Cat. no. 439454, Nunc A/S, Kamstrup, Denmark) were coated with rabbit IgG antibodies to human immunoglobulin (Cat. no. A 107, Dakopatts, Copenhagen, Denmark) diluted 1/10.000 in 0.1 M bicarbonate, pH 9.6. Coated wells were washed with PBS-Tween (phosphate buffered saline-0.05% Tween 20), and incubated for 2 hours at room temperature with supernatants diluted 1: 10 in PBS-Tween. The wells were again washed and incubated for 1 hour at room temperature with either AP (alkaline phosphatase) labelled rabbit anti-human IgG or anti-IgM (Cat. no. D335 and D337, Dakopatts) diluted 1:3000 in PBS-Tween. After washing the substrate, PNPP (paranitrophenylphosphate) was added at I mg/ml 10% diethanolamine, 1 mM MgC12, pH 9.6. Optical density was measured at 405 nm after development for 1 hour at 37 "C. Standard curves for quantification were constructed using IgM (Cappel) or IgG (Kabi AB, Stockholm, Sweden). Cells from Ig positive wells were transferred to 24-well macroplates (Cat. no. 142982, Nunc A/S), and further analyzed for the production of anti-tumor antibody. ELISA for antibody activity. Supernatants from wells containing growing cells were assayed by ELISA for reactivity towards the following cancer cells: Colon 137, Colon 138 (obtained from Dr P. Ebbesen, The Cancer Research Institute, Arhus, Denmark), and COLO 201 (ATCC). These cell lines are derived from human colon adenocarcinomas. ELISA was camed out after glutaraldehyde fixation (0.17% glutaraldehyd in PBS) of the cells onto microplates (38). Development was with AP anti-human IgG or IgM and as described above. Immunohistochemical analyses. Supernatants collected 2 to 4 weeks after fusion with 729-HF2 were screened on frozen sections of colorectal adenocarcinoma and some on normal tonsilar tissue. The frozen blocks were cut into 5 pm sections, air dried overnight at room temperature, fixed in acetone for 10 min, washed in PBS-Tween and preincubated with Fab' rabbit- anti-human IgM or anti-IgG for 4 hours at 4 "C in a humidified chamber to block endogenous immunoglobulin (30). After washing, the sections were incubated overnight at 4 "Cwith undiluted supernatants. The slides were washed and incubated for 1/2 hour at 4 "C with HRP (horse-radish peroxidase) (BoehringerMannheim, FRG) labelled rabbit anti-human IgM or anti-IgG prepared from the same rabbit anti-human Ig
BORUP-CHRISTENSEN et al.
antibody as used for the preparation of Fab’ fragments (30). Four monoclonal antibodies were concentrated by precipitation with 2 M ammonium sulphate and analyzed on different cancer and normal tissues using the described technique. Immunocytochernical analyses. Culture supernatants were analyzed on formol-acetone fixed cell smears ( 5 ) prepared from human tumor cell lines (Colon 137 and COLO 201 (colon adenocarcinoma cell lines), MCF-7 (mamma carcinoma cell line, ATCC), HUTU-80 (duodenal adenocarcinoma cell line, ATCC), EB-2 (Burkitt’s lymphoma cell line, ATCC) and HU 373 (malignant melanoma cell line, kindly provided by Dr I. Nielsen, The Finsen Institute, Copenhagen), and human blood leucocytes. SDS-PAGE and Western blotting. Colon cancer cells at lo7 cells per ml PBS containing enzyme inhibitors (iodoacetamid, tranexamic acid (Cyclocapron, KABI), ethylene diamine tetraacetic acid (EDTA) and pehnylmethylsulfonylfluorid (PMSF), all at I mM) were mixed with an equal volume of sample buffer (2% SDS, 4 M urea, 10 mM iodoacetamid) and incubated at ambient temperature for 15 min before PAGE on 5 to 209’0 gradient gels. The proteins were transferred onto nitrocellulose sheets as described (40). The nitrocellulose sheets were cut into 3 mm stripes. Proteins were stained by colloidal gold (39). For immuno-staining, blots were quenched in TBS, 0.1% Tween-20, for 10 min and incubated overnight at 4 “C with 2 ml of hybridoma supernatant, followed by three washes in TBS (140 mM NaC1, 10 mM Tris/HCI, 10 mM NaN,, pH 7.4), 0.019’0 Tween. Bound monoclonal antibody was detected by incubation with AP-rabbit anti-human IgM (Sigma), 1/1000 in TBS, 0.019’0Tween for 2 hours. The blots were washed with PBS and fixed by incubation for 15 min with 0.2% glutaraldehyde in PBS. The glutaraldehyde was blocked with 0.1 M ethanolamine buffer, pH 9.0. The alkaline phosphatase was visualized by incubation for 1 h at 37 “C with the substrate, nitro blue tetrazolium and 5-bromo-4chloroindoxyl phosphate diluted in 0. I M ethanolamine buffer, pH 9.0 (3). The glutaraldehyde fixation was introduced to eliminate the risk of eluting monoclonal antibody at the basic pH of the substrate. The M, was
calculated from the mobility of the following prestained molecular weight markers (Sigma, Cat. no. SDS7B):triosephosphate isomerase (26.6K); lactic dehydrogenase (36.5K); fumarase (48.5K); pyruvate kinase (58K); fructose-6-phosphate kinase (84K); beta-galactosidase( I I6K). Flow cytometric DNA analysis. The DNA distribution of nuclear DNA was analyzed by flow cytometry on hybridoma and parental cultures, as well as on normal male blood lymphocytes, Raji cells (Burkitt’s lymphoma, ATCC CCL-86) and BJAB cells (EBNA-negative Burkitt’s lymphoma) (28). The nuclei were prepared and stained by treatment of the cells with 1% non-ionic detergent (NP-40) and propidium iodide (50 pg/ml) in a hypotonic, neutral solution containing RNAse (25). Chicken and trout erythrocytes were used as internal DNA standards. The fluorescenceat above 580 nm of at least lo4nuclei was analyzed in a Becton Dickinson FACS IV cell sorter using 488 nm for excitation (CV 2.4-4.79’0). From the fluorescence histogram the DNA index (DI) and the cell cycle distribution (G,, S, and G2M fractions) were estimated by maximum likelihood, assuming normally distributed measurement error and uniform distribution of DNA in the S-phase. The DI scale was corrected for non-linearity of the peak fluorescence intensity, so that a DI of 2 corresponded to tetraploid DNA content (10, 42). The sample DI was calculated by dividing the mean of the G, peak by the mean value of lymphocytes relative to the trout internal standard. DI confidence limits for lymphocytes were & 0.036 at the 19‘0 level (25). The difference in DI estimates between duplicate frozen samples, stained and analyzed at different days, was less than 0.04.
Clonal outgrowth. Twenty-five fusions were performed between lymphocytes from mesenterk lymph nodes from patients with colorectal cancer and two different human B-lymphoblastoid cell lines. There was no difference in clonal outgrowth.
TABLE 1. Results of fusion with LICR-LON-HMy-2 Fusion no.
Wells with hybridoma
Supernatants reactive with colon cancer cells in ELISA
480 480 480 480 480 288
6 10 0 153 124 I
0 3 0 8 15 0
HUMAN MONOCLONAL ANTIBODIES TO EDORECTAL CANCER
TABLE 2. Results of fusion with WI-LZ-729-HF2
Wells seeded Wells with hybridomas Wells containing IgM Wells containing IgG Wells containing IgM and IgG
Fusion 1-10 (HAT)
Fusion 1 1-19 (AH)
2328 458 134 51 58
3936 1787 290 21 41
Supernatants reactive with colon cancer cells in ELISA
44 (IgM) 5
Supernatants reactive with colon cancer in immunohistochemistry
29 (IgM) 14
The number of clones obtained with HMy-2 (Table 1 ) corresponds to 32 clones per lo7 lymphocytes used while 729-HF2 gave 34 (Table 2). By changing selection medium from HAT to AH the clonal outgrowth using 729-HF2 as fusion partner was 44 clones per lo7 lymphocytes. Clone stability, Ig secretion and primary screeningqf antibodies. HMy-2 secrete IgG, (kappa) and, using this cell line as fusion partner, all wells with growing cells were found to contain Ig. The primary supernatants were not tested with class specific assays. Supernatants from 26 out of 294 hybridomas reacted with colon cancer cells in ELISA. Only one of these 26 hybridomas (D4213) was stable after cloning and subcloning. It produces both IgM (kappa), IgM (lambda), IgG (kappa) and a small amount of IgG (lambda). Only the IgM showed binding to colon cancer cells in immunocytochemical and immunohistochemical analyses. Using 729-HF2 as fusion partner and HAT as selection medium, IgM, IG or both were detected in 53% of the wells with growing clones. By changing to AH medium for selection, Ig was detected in only 19% of the wells with growing clones. Only the IgM producing hybridomas were stable, the IgG production ceased within a few weeks.
The reactivity against colorectal cancer of monoclonal antibodies obtained after fusion with 729HF2 was estimated in to different analyses (Table 3). Out of 194 hybridomas producing antibodies reacting with colorectal cancer, 20 have until now been cloned. They all produce IgM. Despite attempts, we have not succeeded in cloning hybrids producing IgG. It should be noted that most of the antibodies were positive in only one of the two test systems. Despite successful cloning many of the hybridomas are very unstable and low secretors, requiring cloning and recloning with short intervals. Four hybridomas (G4 146, Fll348, B9 165 and D4213) which turned out to be stable, have been in culture for 3-4 years and produce from 0.5-5 pg IgM per ml, were chosen for further screening. Flow cytometric DNA analysis. Every sample contained a single cell populaiton with an S-phase fraction above 25% indicating rapid growth. The hyperdiploid DNA content of the parental cell lines 729-HF2 (DI 1.13-1.16) and HMy-2 (DI 1.09) was similar to that of other B lymphoblastoid cell lines: EBNA positive Raji cells (DI 1.15) and EBNA negative BJAB cells (DI 1.17). The DNA contents of the three hybridoma lines F11348 (DI 2.10), G4146 (DI 2.19) and D4213
TABLE 3. Reactivity pattern on hybridoma supernatants obtained by fusion with WI-L2-729-HF2 Immunoglobulin
Positive only in ELISA on tumor cells
Positive only on colon cancer tissue
Positive both in ELISA and on cancer tissue
BORUP-CHRISTENSEN ef al
TABLE 4. Reactivity of five human monoclonal IgM antibodies on cell smears
Colon carcinoma: Colon carcinoma: Mamma carcinoma: Duodenum carcinom: Burkitt lymphoma: Melanoma: Blood leucocytes:
Colon 137 COLO 201 MCF-7 HUTU-80 EB-2 HU 373 PBL
Myeloma IgM (Cappel)
+ + +
+ + + + + + +
+ + + + + +
Fig. 1. Immunocytochemical staining of colon I37 (colon adenocarcinoma) and HUTU-80 (duodenal adenocarcinoma). B9165 shows binding to the periphery (arrows) of some of the colon 137 cells (A) and no binding to HUTU-80 (B). D4213 shows diffuse staining of colon 137 cells (C) and no reaction with HUTU-80 (not shown). G4 146 shows reaction to the periphery of both colon 137 (D) and HUTU-80 (not shown).
HUMAN MONOCLONAL ANTIBODIES To EDORECTAL CANCER
TABLE 5. Reactivity of four human monoclonal IgM antibodies onfrozen, acetone-fixedcoloncancer tissues (C 1-15) and the corresponding normal colon epithelium (N 1-15)
CI c 2
c5 C6 c 7 C8 c9
c 10 c 12
C 13 C 14 C 15
5 pg/ML 0.5 pg/ml
10 pg/ml 0.5 pg/ml
5 pg/ml 2 pg/ml
10 pg/ml 2 pg/ml
+++' +++ +++ +++ +++ +++
+++ +++ +++ +++ +++
++ + + + + ++ + ++ + + ++ + ++ ++ +
+ + + +
+++ +++ +++ +++ +++ +++ +++ +++
Colon cancer stroma -
+ ++ ++ + ++ + +
+ ++ ++ ++ +++ + +
+++ +++ +++ +++
+ + + + -
+++ ++ ++ + +++ ++ 'Strong reaction: +++; Moderate reaction: ++; Weak reaction: +; No reaction: -. /'Focal binding: most cells stained f; few cells stained T . N 1-15
Human myeloma IgM (Cappel) 10 pg/mI
+++ +++ +++ +++
+++ ++ +++ ++
-/+++ -/+++ -/+
TABLE 6. Reactivity of four human monoclonal IgM antibodies on frozen, acetone-fixed cancer and normal tissues
B9165 5 Piid ml
Renal carcinoma Renal tubules Renal globuli Lung carcinoma Alveoli Bronchial epithelium Endothelium Smooth muscles Ovarial carcinoma Ovarial epithelium Ovarial stroma Mammary carcinoma Mammary ductuli Mammary lobuli Tonsillar tissue Malingant melanoma Epidermis astrong reaction:
FI 1348 10 pg/
++ ++ ++ ++ ++ ++ +++ +++ ++ ++ ++ ++ +++ +++ ++ ++ +++
0.5 pg/ ml
5 pg/ ml
2 pg/ ml
2 pg/ ml
Human myeloma IgM (Cappel)
10 pg/ ml
+++; Moderate reaction: ++; Weak reaction: -. 679
BORUP-CHRISTENSEN et al.
Fig. 2. Immunohistochemical staining of frozen, acetone-fixed colon cancer tissue with human IgM (Cappel) (A) and B9 165 (B), and D4213 (C). Also shown is ovarian cancer tissue with B9165 (D).
DI 2.18; late passage: DI 2.15) were all hypertetraploid, whereas in the B9 165 hybridoma the DI decreased significantly from 1.80 in an early passage to 1.67 in the latest passage. Immunocytochemical analyses. The four antibodies were further analyzed by the immunocytochemical technique on a range of cancer cell lines and normal human leucocytes (Table 4). Two of the monoclonal antibodies showed selective reactivity in this analysis. B9165 which reacts with colon cancer and mammae cancer cells appears to bind to the periphery of the cells but not all the cells are stained. D42 13, which binds to colon cancer and melanoma, on the other hand, shows diffuse 680
staining over the entire cell and all the cells are stained. F 1 1 348 and G4 146 show binding located to the periphery of all the cells analyzed. Some of the staining reactions are shown in Fig. 1. Immunohistochemical analyses. The reaction pattern of the four monoclonal antibodies on different tumors and normal tissues analyzed on frozen, acetone-fixed tissue sections is shown in Table 5 and 6. Common for all our IgM antibodies is that they bind to normal colon epithelium with a diffuse staining pattern. Similar staining was seen when applying normal human polyclonal IgM or different myeloma IgM preparations. This apparently
HUMAN MONOCLONAL ANTIBODIES TO EDORECTAL CANCER
1 2 3 4 5 6 7 Fig 3. Analysis of the reactivity of antibodies by Western Blotting. Extract of colon cancer cells was separated by SDS-PAGE and transferred to nitrocellulose. Lane 1 shows protein staining with colloidal gold. Lanes 2 to 7 show the staining after incubation with hybridoma supernatant and AP-anti-IgM antibody. Lane2:F11348; Lane3:G4146;Lane4:B9165;Lane5: D42 13; Lane 6: Myeloma IgM (Cappel); Lane 7: control with cell culture medium.
non-specific binding of IgM to colon epithelium was not observed when using formaldehyde-fixed tissues instead of frozen, acetone-fixed tissues. The same unspecific binding was found to bronchial epithelium. Myeloma IgM (Cappel) showed binding to 6/15 colon cancers, but the binding was restricted to a very few epithelial cells (Fig. 2A), while the antibodies all have a more diffuse staining pattern (Fig. 2B and 2C). Binding of B9165 (and G4 146 and Fll348 not shown) was restricted to colon cancer cells, while D42 13 bound to both cancer cells and the surrounding stroma. Fig. 2D shows reactivity of B9165 to ovarial cancer cells
with no staining of cancer stroma. Three of the monoclonal antibodies were reactive against all 15 colon cancers when analyzed at 5- 10 pg antibody/ml. Reactivity against other tumors and normal tissues was, however, also seen. F11348 reacts with all tissues tested, while B9 165 shows a reaction with malignant and normal tissues from epithelial origin. D42 13 also reacts with a range of epithelial tumors and normal tissues but not with mammae carcinomas. In addition, D42 13 shows binding to malignant melanoma, smooth muscles and endothelium. G4146 shows besides a reaction with 14/15 colorectal concer binding to malignant melanoma. This antibody, in addition, reacts with smooth muscles and with renal tubules and endothelium. When analyzed at a lower concentration, all 4 monoclonal antibodies showed more selective reactivity to cancer (Tables 5 and 6). The reactions with normal tissue components were based on observations with biopsies from a large number of individuals, except for renal tissue, lung tissue, ovarian tissue and epidermis, where only a few biopsies were examined in each case. Antigen analyses. Attempts were made to identify the target antigens for the four human monoclonal antibodies. Extracts from Colon 137 cells were treated with SDS and separated on PAGE, followed by blotting onto nitrocellulose sheets (Fig. 3). One hybridoma supernatant (F11348) reacts with several components. G4 146 binds to a double component with M, about 59K and 6 1K, while B9 165 reacts with a component with M, of about 43K. D4213 showed no reaction in this analysis (Fig. 3, lane 5).
DISCUSSION The fundamental issue in cancer immunology is to establish to what extent the immune system recognizes spontaneously occurring autologous tumors. In humans this question has recently been addressed using methods to immortalize lymphocytes. This has resulted in human monoclonal antibodies with apparently selective reactivity against various tumors such as myeloid leukaemia (2), lung cancer ( 17), prostate cancer (26), malignant melanoma (20), and colorectal cancer ( 15).In extensive studies reported by Houghton et al. ( 18) 68 1
BORUP-CHRISTENSEN et a/
and Cote et al. ( 1 1) lymphocytes from draining lymph nodes of melanoma lesions were used as the source of antigen-primed lymphocytes. Most of the resulting human monoclonal antibodies were found to react with intra-cellular structures that were also present in normal cells. Haspel et al. ( 15) described the successful production of human clones producing antibodies which recognized antigenic determinants preferentially expressed on the cell surface of colorectal cancer cells. They used peripheral blood lymphocytes from colorectal cancer patients immunized with autologous tumor cells. The claimed surface reactivity was not formally proven and later results showed that cytoplasmic molecules are labelled (27). We have used human-human hybridoma technology to study the recognition of tumor antigens by the humoral immune system in patients with colorectal cancer. The hybridization was performed using two different human B-lymphoblastoid cell lines, and lymphocytes from tumor-draining lymph nodes. Only a fraction (27%) of the growing hybridomas (2245) obtained after fusion with 729-HF2 were found to produce immunoglobulin. About 33% of these (194) showed a reaction with cancer cells. For various technical reasons (loss of immunoglobulin production, loss of viability, etc.) not all of our hybridomas were analyzed for reactivity against differentiation antigens, i.e. antigens expressed in detectable amounts on some but not all cell types. Out of the 194 antibody-producing hybridomas, it was possible to test 37 for tissue specificity by immunohistochemical analysis. By this method, 19 were found to react with colon cancer but not with tonsilar tissue. None of the antibodies have shown reactivity restricted to autologous cancer cells, that is type I reactivity, but should rather be classified as type I1 ( 18). In comparison, others have found by similar techniques that between 15 and 2OYo of immunoglobulin-producing clones show reactivity with cancer tissue (1 5, 37). In our study, approximately 29% of the Ig positive primary supernatants contained IgG. Ten percent of these showed binding to cancer cells. The frequency of IgM positive supernatants was three-fold higher (some supernatants contained both IgG and IgM), and 3490 of the hybridoma IgM showed binding to cancer cells. The higher frequency of IgM than of IgG producers is in 682
agreement with the prelavence of IgM B-cells (7). In the binding assays, IgM will have an advantage over IgG due to the polyvalency which may confer a sufficient avidity for binding even if the individual binding sites have low affinity. This may account for the observed higher frequency of monoclonal IgM reacting with cancer. Instability of human IgG-producing hybridomas was also found by Kan-Mitchell et al. (20). Other groups (1 8, 37) have succeeded in obtaining human IgG producing hybridomas which appear to be stable. Four of our IgM-producing hybridomas have been cloned and further analyzed by flow cytometric analysis of nuclear DNA content. The applied parental cell lines (729-HF2 and HMy-2) as well as other B-lymphoblastoid cell lines (Raji and BJAB) have a hyperdiploid DNA content in the same range (DI 1.10-1.20) as reported for acute lymphoblastic leukemias (8). The hypertetraploid DNA content in three of the hybridoma lines, F11348, G4 146 and D42 13 is consistent with the fusion between a diploid lymphocyte and a hyperdiploid B-lymphoblastoid cell, resulting in a hybridoma with stable karyotype. The B9165 hybridoma was probably formed in the same way, but, in contrast to the other hybridomas, it has gradually lost genetic material. This is indicated by a significant decrease in DI from early to late passages. Immunocytochemical staining on different cancer cell lines and normal human leucocytes showed distinct reaction patterns for each of the antibodies. B9 165 and D42 13 clearly reacted with differentiation antigens expressed only by some of the tumor cell lines. All the antibodies showed binding to sections of frozen normal colon epithelium. Similar binding was also found with the two control IgM preparations, i.e. normal human IgM and myeloma IgM from Cappel. Controls without application of IgM showed no binding of the enzyme conjugated anti-IgM antibody. Kan-Mitchell et al. (20) suggest that such binding of IgM to epithelial colonic tissue is non-specific. Myeloma IgM also showed binding to 6/15 colon cancer tested, but this binding was only focally located to the cancer cells compared to diffuse binding obtained with the human monoclonal antibodies. When tested at 5-10 pg/ml the monoclonal antibodies showed diffuse binding to nearly all colon cancer analyzed. In addition, they also reacted with other tumors and in varying degrees
HUMAN MONOCLONAL ANTIBODIES TO EDORECTAL CANCER
with normal tissues. When the antibodies were analyzed in a lower concentration they showed a more selective reactivity, suggesting differences in the amount or nature of the antigen in normal and malignant tissues. The reactivity of the monoclonal antibodies was further analyzed by incubation on nitrocellulose blots of SDS-PAGE separated tumor cell extract. One of the antibodies(Fll348)reacted with several protein bands, while two of the antibodies (G4146 and B9165) each identified distinct molecules. The resutls obtained demonstrate the successful application of hybridoma technology to produce human monoclonal antibodies with reactivity to differentiation antigens. One of the human monoclonal antibodies (B9 165)has by electron microscopy and isoelectric focusing shown binding to colon cancer tissue but not to the corresponding normal colon epithelium (data not shown). The hybridoma cell line producing this antibody is very stable and secretes about 5 pg IgM per ml. Based on these findings we have selected B9 165 antibody for further studies concerning its possible ability to localize tumors in patinets with colorectal cancer. ~
Supported by grants from The Danish Cancer Society and The Danish Medical Research Council.
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