Epitopes of carcinoembryonic antigen (CEA) defined by monoclonal antibodies

Of 15 anti-CEA monoclonal antibodies, the first 8 were reactive only with CEA, while the remaining 7 antibodies reacted with epitopes commonly expressed on CEA and the normal cross-reacting antigen, NCA. Separate and distinct, conformation-dependent (i.e. susceptible to reduction and alkylation), CEA-associated epitopes were identified using antibodies 1, 2 and 3. Antibodies 4 to 7 defined a series of conformation-independent epitopes which were topographically closely related on the CEA molecule. Antibody number 8 reacted with a separate determinant found on CEA but not NCA, and this also was resistant to reduction and alkylation. Antibody number 9 defined an epitope which was commonly expressed on CEA and NCA. This epitope was conformation-dependent and was the most sensitive to NaIO4. The remaining antibodies, 10 to 15, which also reacted with CEA and NCA, defined an immunodominant region of these molecules since the 6 epitopes were clearly closely related, but not necessarily identical. The findings presented establish a rational basis for the selection of combinations of anti-CEA antibodies for diagnostic and therapeutic purposes.     

Clinical application of carcinoembryonic antigen (CEA) monoclonal antibodies (McAbs)

CEA McAbs, recognizing three different epitopes on CEA molecules, were used to measure serum CEA level in cancer patients by enzyme-immunoassay (EIA). The results, as compared with those using polyclonal antibodies, indicated that the positive rate was higher while the false positivity was lower. Immunohistochemistry of tumour sections showed that the CEA McAbs are bonded to 80-90% of gastrointestinal cancers. Although the normal colon epithelium occasionally reacted with CEA McAbs, other normal tissues did not. After in vivo administration of radio-labeled CEA McAbs to nude mice xenograft with human colon cancer, the radio-isotope was found to be concentrated preferentially in the tumour. The ratio of tumour and normal tissue was 3.6-11.8 after 48 hours following administration. Thus, the CEA McAbs can be used clinically not only for serum CEA determination but also for diagnostic imaging.     

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA)

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.     

Immunohistochemical localization and molecular characteristics of three monoclonal antibody-defined epitopes detectable on carcinoembryonic antigen (CEA)

Three murine monoclonal antibodies (MAbs) reactive to different epitopes on CEA were selected according to their ability to bind to various human tissue sections. The most selective MAb, BW 431/31, bound to the majority of colon carcinomas but only faintly to normal colon mucosa. In addition to the tissues stained by MAb BW 431/31, MAb BW 250/183 reacted with granulocytes, colon mucosa and faintly with pancreatic ducts. The third MAb, BW 374/14, reacted with granulocytes, colon mucosa, strongly with pancreatic ducts and with alveolar and bronchial epithelium. The antigenic determinants recognized by the 3 MAbs in human tissue sections were resistant to formaldehyde fixation and paraffin embedding as well as to periodic acid oxidation and neuraminidase treatment. The last two treatments suggest that the epitopes are protein in nature. Using MAb affinity chromatography, 3 antigen preparations were isolated from a human colon carcinoma xenograft with an approximate molecular weight of 180 kd. These preparations were shown to bear the epitopes from each of the MAbs and from a polyclonal antiserum specific for purified CEA. Furthermore, the ability of MAb BW 431/31 to localize its antigenic determinant in vivo on a human colon carcinoma xenograft is evaluated and its possible application in the patient is suggested.     

Evaluation of the measurement of serum carcinoembryonic antigen (CEA) levels using monoclonal antibody

We measured serum carcinoembryonic antigen (CEA) levels in 164 cancer patients, 153 patients with benign diseases and 45 healthy controls using monoclonal antibody and compared CEA levels by monoclonal antibody (m-CEA) with those by polyclonal antibody (p-CEA). There was a good correlation between m-CEA and p-CEA, especially in cancer patients. The positively of m-CEA was almost the same as that of p-CEA in cancer patients. But, false-positive cases by m-CEA were less common than by p-CEA in non-cancerous patients. Thus, the measurement of m-CEA was not less useful than that of p-CEA.     

Serologic mapping and biochemical characterization of the carcinoembryonic antigen epitopes using fourteen distinct monoclonal antibodies

A series of 14 monoclonal anti-carcinoembryonic antigen (CEA, Mr 180,000) antibodies (MAbs) that show a strong degree of selective reactivity for human colon carcinomas versus normal adult tissues were used to construct a serological map of the CEA molecule. The MAbs were generated using extracts of colon carcinomas as immunogen and are thus given a COL designation. None of the 14 COL-MAbs tested were reactive with purified non-specific cross-reacting antigen (NCA, Mr 55,000) from normal lung, although some showed reactivity to human granulocytes. All the COL-MAbs tested were reactive with normal fecal antigen-2 (NFA-2, Mr 170,000); however, many of the COL-MAbs demonstrated a higher affinity constant to CEA than to NFA-2. Cross-competition radioimmunoassays classified the 14 COL-MAbs into 5 groups. The chemical nature of the COL-binding domains was tested using chemically or enzymatically treated CEA; all reacted with periodate-treated CEA and deglycosylated CEA, indicating that the COL-reactive epitopes appear to be of a proteinaceous nature. Heat treatment, reduction, alkylation, pepsin digestion or pronase treatment of CEA, however, gave differential results with respect to COL binding. Antibody titration experiments were carried out to define differential reactivities to colorectal carcinoma versus NCA-containing granulocyte extracts; these results were compared with results obtained using several anti-CEA MAbs that have been used in clinical trials. Granulocyte binding and biochemical studies showed that the COL MAbs may distinguish as many as 7 to 10 CEA epitopes.     

Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.

The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.     

Conformational epitopes specific to carcinoembryonic antigen defined by monoclonal antibodies raised against colon cancer xenografts

Four monoclonal antibodies (MoAbs) reactive with carcinoembryonic antigen (CEA) were obtained by hybridizing mouse myeloma cells (P3-X63-Ag8-U1) with spleen cells from nude mice (BALB/c, nu/nu) that had rejected transplanted human colonic adenocarcinomas Co-3 and Co-4 following intraperitoneal injection of spleen cells from immunocompetent mice (BALB/c). By solid-phase RIA with purified CEA and its related antigens, NCC-CO-413 (IgG2a, kappa) was shown to react with NCA and BGP-I as well as with CEA, whereas the reactivities of three other MoAbs, NCC-CO-308 (IgG1, kappa), -432 (IgG1 lambda), and -411 (IgG1, kappa) were limited to CEA. Immunohistochemical reactivities of these MoAbs to colonic carcinomas, granulocytes, and liver bile canaliculi on acetone-fixed paraffin-embedded sections ("AMeX" sections) confirmed the specificities of these MoAbs shown by the solid-phase RIA. By competition solid-phase RIA, the epitopes recognized by NCC-CO-308 and -432 were shown to be shared or located close to each other, whereas the other MoAbs were shown to recognize different epitopes. Thus, two epitopes specific to CEA and one shared by NCA and BGP-I as well as CEA were identified. Furthermore, reactivities of MoAbs with the two CEA-specific epitopes were easily abolished by heat denaturation or reduction of CEA, as revealed by solid-phase RIA and SDS-PAGE-immunoblotting, indicating that these two CEA-specific epitopes are based on the conformational structure of the CEA molecule.     

Mucin-like carcinoma-associated antigen defined by three monoclonal antibodies against different epitopes

Three monoclonal antibodies [MAb], b-8, b-12, and b-15, have previously been shown to react with mammary carcinomas and with a restricted set of cells in normal human tissues [C. St?hli et al., Experientia (Basel), 41: 1377-1381, 1985; H. R. Zenklusen et al., Virchows Arch. Abt. A Pathol. Anat., 413: 3-10, 1988]. They are shown here to recognize the same high molecular weight acid soluble glycoprotein antigen. Lectin binding, biolabeling, and deglycosylation experiments demonstrate that it contains O-linked carbohydrate side chains with sialic acid and hexoses including fucose, galactose, and/or galactosamine but little if any mannose. These properties, typical of mucin-like glycoproteins, agree with the antigen expression on mucin-secreting epithelial surfaces (H. R. Zenklusen et al., Virchows Arch. Abt. A Pathol. Anat., 413:3-10, 1988). The antigen is thus named mucin-like carcinoma-associated antigen (MCA). The three MAb are shown to bind to three different epitopes on MCA. Two of these epitopes (MCA-b-8 and MCA-b-15) are O-linked carbohydrates, and one (MCA-b-15) contains sialic acid. The epitope MCA-b-12 is of peptide nature. Of various two-site sandwich enzyme immunoassays composed of different combinations of the three MAb, the one with MAb b-12 in both positions is selected for a serum assay. Analyses of tumor patients' sera demonstrate that this MCA enzyme immunoassay can be of use as a tumor marker assay for mammary carcinomas. The parameter MCA enzyme immunoassay is shown to differ from other parameters described in the literature.     

Factors influencing variability of localisation of antibodies to carcinoembryonic antigen (CEA) in patients with colorectal carcinoma--implications for radioimmunotherapy.

Tumour localisation of anti-tumour antibodies varies greatly between patients. Factors which may be responsible for this have been investigated in 56 patients with colorectal carcinoma with a view to improving radioimmunotherapy. Thirty-seven to seventy-four MBq of 125-I labelled mouse monoclonal antibody to CEA, was given intravenously and tumour resected 70-480 h later. Percentage injected activity kg-1 (% inj.act kg-1) in tumour, was inversely correlated with the time interval between injection and operation (P = 0.004). To assess the influence of other parameters on localisation, patients were divided into two time groups according to time interval between injection and operation, 70-120 h (n = 33) and 144-480 h (n = 23). In neither group was there a significant correlation of % inj.act kg-1 with time. The % inj.act kg-1 in tumour showed a significant correlation with that in the blood for both groups (P = 0.005 and P = 0.01). There was no significant correlation for either time group between % inj.act kg-1 in tumour and serum CEA values, the per cent of tumour cells positive for CEA and vascularity. Tumour to blood ratios varied considerably (range 0.3-28.5:1) suggesting that factors other than time and persistence of activity in the blood contribute to efficient targeting. Tumour to blood ratios were inversely correlated with % inj.act kg-1 in blood for the 70-120 h group (P = 0.007), and were positively correlated with % inj.act kg-1 in tumour (P = 0.012). Autoradiography showed that antibody localised predominantly on tumour cells but was distributed heterogeneously, was not solely related to the expression of antigen and in some cases accumulated in necrotic more than viable areas of tumour. Penetration of antibody into malignant acinar structures was poor and CEA-positive cells closer to the blood supply were targeted to a greater extent than distant cells. Preoperative administration of radiolabelled antibody to CEA may be helpful in selecting patients with favourable localisation for radioimmunotherapy.     

Immunological heterogeneity of carcinoembryonic antigen: antigenic determinants on carcinoembryonic antigen distinguished by monoclonal antibodies

Murine monoclonal antibodies against carcinoembryonic antigen (CEA) derived from a colonic tumor were analyzed by radioimmunoassay for reactivity with CEA and the CEA-related antigens, meconium antigen (MA) and nonspecific cross-reacting antigen. Antibody-antigen binding profiles revealed three classes of hybridomas. The Class I antibody, NP-1, recognized an epitope shared among all three antigens, and its affinity for CEA and MA was high but low for nonspecific cross-reacting antigen. The Class II antibodies reacted with sites shared between CEA and MA, while those of the Class III type only bound CEA. The Class III antibody, NP-4, bound less than 50% of the CEA molecules recognized by goat specific anti-CEA antibody and the other classes of monoclonal antibodies. Two Class II antibodies, NP-2 and NP-3, bound similar amounts of CEA and MA with moderate but different affinities for CEA. Studies using labeled monoclonal antibodies for CEA epitope blocking revealed that NP-2 and NP-3 recognize two separate epitopes on CEA within the Class II category. Thus, monoclonal antibodies to CEA can differentiate at least four antigenic sites on colonic cancer CEA. One is shared by CEA, MA, and nonspecific cross-reacting antigen; two others are shared by CEA with MA; and a fourth appears specific for a subpopulation of CEA molecules.     

A monkey antigen crossreacting with carcinoembryonic antigen, CEA.

Normal monkey tissues were found to contain an antigen which crossreacts immunologically with the carcinoembryonic antigen (CEA) of the human digestive tract. The monkey antigen reacted with complete or partial identity to the normal crossreacting antigen (NCA) in humans when tested in immunodiffusion against anti-CEA or anti-NCA. Extracts of monkey tissues inhibited in radioimmunoassays measuring human NCA. It is possible that monkey foetuses and colonic tumours contain CEA.     

Clinical use of carcinoembryonic antigen (CEA) determination]

The carcinoembryonic antigen (CEA) is a tumor marker defined by specific heterologous antisera. Elevated levels of circulating CEA have been detected by radioimmunoassay in 20-90 per cent of cases of colorectal carcinomas depending on the degree of tumor spread. The fact that elevation of CEA level can also be observed in other types of carcinomas and in several non malignant conditions greatly limit the value of the CEA test for the early diagnosis of colorectal carcinoma. Thus, the CEA assay should not be used as a screening test for cancer. Repeated CEA measurements, however, appear to be of importance for the evaluation of tumor resection and the detection of tumor recurrence. The only localized tumors known to produce elevation of CEA above the levels observed in non malignant diseases are carcinomas of the large bowel and the pancreas. In carcinomas derived from other organs a marked increase of CEA level is always associated with the presence of distant metastasis. Therefore at the present time the clinical applications of the CEA radioimmunoassay should be limited to the differential diagnosis of patients with suspicion of primary colorectal or pancreatic carcinoma, to the detection of distant metastasis in other types of carcinomas and to the post operative follow up of patients who had elevated levels of CEA before surgery. Well-controlled studies are still needed to determine if therapeutic decisions based on CEA results can lead to improved survival.     

Tumor specificity of monoclonal antibodies to carcinoembryonic antigen. Immunohistochemical analysis

The tumor specificity of twelve different monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) was assessed by immunohistochemistry. The Mabs had previously been classified into three specificity groups (I-III) on the basis of their reactivity with purified CEA-related antigens by ELISA. Mabs belonging to specificity group III (n = 4) did not cross-react with any CEA-related antigen, including normal cross-reactive antigen of 160 kD molecular weight (NCA-160 = meconium antigen). All Mabs, except one, gave positive immunohistochemical staining of 75-100% of individual tissue samples of colorectal carcinomas and gastric adenocarcinomas. However, when tested against different normal adult tissues, the Mabs displayed marked differences in reactivity. Group III Mabs stained normal colon epithelium, but not parenchymal cells in other organs or, with one exception, cells belonging to the granulocyte and/or macrophage series. Group I and II Mabs, in contrast, stained parenchymal cells in normal colon, submandibular salivary gland, placenta, and pancreas (group I Mab only). They also stained infiltrating and circulating granulocytes and/or macrophages. Lack of cross-reactivity with NCA-160 is the single-best criterion for selecting anti-CEA Mabs with a high degree of tumor specificity. To ensure tumor specificity, CEA-positive, NCA-160-negative Mabs should be checked by immunohistochemistry against cryostat sections of colorectal carcinoma, normal pancreas, submandibular salivary gland, spleen, and liver and for reactivity against circulating granulocytes.     

Faecal carcinoembryonic antigen (CEA) in patients with large bowel cancer

The possibility that faecal CEA may be a more useful measurement than serum CEA for the detection of large bowel cancer has received very little attention. For this reason faecal CEA was measured before and after tumour resection in colorectal cancer patients and in a variety of control subjects. CEA was extracted from faeces by a new method with 3M KCl and assayed by an EIA technique utilising two monoclonal antibodies. Mean +/- SE faecal CEA in 32 cancer patients was 4.15 +/- 1.17 micrograms/g preoperatively. Values were not related to either stage of disease or serum CEA and they fell to 0.83 +/- 0.34 micrograms/g (n = 20) following tumour resection (P less than 0.05). Mean +/- SE faecal CEA in 34 control patients with no known colorectal disease was 0.94 +/- 0.49 micrograms/g which was significantly lower than in the cancer patients (P less than 0.05). Furthermore faecal CEA in 25 patients with non malignant colorectal disease was 1.44 +/- 0.63 micrograms/g which again was significantly lower than that in the cancer patients. It is concluded that as CEA is present in the faeces of the majority of colorectal cancer patients even at early stages of the disease its measurement here may be more useful for the detection of large bowel cancer than that in serum.     

First British standard for carcinoembryonic antigen (CEA).

In 1974, the National Institute for Biological Standards and Control (NIBSC) established the first British Standard for carcinoembryonic antigen (CEA) for use in comparative quantitative assays. The Standard, which was prepared for material processed by the Chester Beatty Research Institute, is in the form of a freeze-dried powder, sealed in all glass ampoules code labelled 73/601 and containing pure dry nitrogen. For practical purposes, each ampoule contains 100 units of CEA activity.     

Characterization of monoclonal antibody CL58 against carcinoembryonic antigen (CEA) and study of its biodistribution

Carcinoembryonic antigen (CEA), which was discovered by Gold and Freedom[1] in 1965, is a highly glycosylated membrane bound protein with a molecular weight of approximately 180kD. CEA is expressed at greatly increased levels in nearly all human colorectal carcinomas, as well as many other malignancies of epithelial origin such as breast, lung and ovarian carcinomas[2-4]. Antibodies are highly specific recognition molecules and the development of McAbs has revolutionized the medical appli…