Determination of the specificities of monoclonal antibodies recognizing members of the CEA family using a panel of transfectants

Carcinoembryonic antigen (CEA), one of the most clinically important tumor markers, is mainly used in the post-surgical surveillance of patients with colorectal carcinomas. CEA belongs to a large protein family, which includes cross-reacting antigens, e.g., non-specific cross-reacting antigens (NCAs) and biliary glycoprotein (BGP) as well as pregnancy-specific glycoproteins (PSGs). The genes encoding these proteins can be subdivided into the CEA and PSG subgroups. The members of the subgroups share antigenic determinants and show high similarity in amino-acid sequences. Their derived secondary structures show them to belong to the Immunoglobulin superfamily. Due to the close relationship of the members of the CEA subgroup, it is very difficult to distinguish between the individual members with MAbs. Here we have used flow cytometric analysis of transfectants expressing individual members of the CEA subgroup as an alternative approach to determine the specificities of 13 MAbs. This allows us to examine the specificities of these antibodies for members of the CEA family, even of those which have not yet been characterized at the protein level. In addition, binding of the MAbs to NCAs expressed by polymorphonuclear cells (PMN) was tested by Western-blot analysis, immunoprecipitation and flow cytometry. Four antibodies bound exclusively to NCA-50/90 and one MAb (80H3) only to NCA-95. MAb 4/3/17 recognizes CEA and BGP on the surface of transfectants and NCA-160 from granulocytes. We assume that NCA-160 is a product of the BGP gene. On granulocytes, which do not express CEA, MAb 4/3/17 is specific for NCA-160 (BGP). Mutual inhibition of the MAbs binding to NCA-50/90 revealed 3 different epitope groups.     

Definition of the expression of the human carcinoembryonic antigen and non-specific cross-reacting antigen in human breast and lung carcinomas

Mouse cell lines transfected with carcinoembryonic antigen (CEA) and with 2 other members of the human CEA gene family, non-specific cross-reacting antigen (NCA) and biliary glycoprotein (BGP), were used to analyze the specificity of several monoclonal antibodies (MAbs). MAbs COL-1 and COL-6 were shown to react with the transfected CEA gene product but not with NCA, confirming previous results. Cells expressing the transfected BGP gene product also failed to react with COL-1 and COL-6. The MAb B6.2 reacted with cells expressing the NCA gene product but not with those expressing CEA or BGP. The MAb B1.1 reacted strongly with the transfected CEA and BGP gene products but only weakly with the NCA gene product. These antibodies were then utilized in the histochemical analysis of a number of primary and secondary breast and lung tumors. The results indicate that a majority of breast and lung tumors express CEA, and nearly all breast and lung tumors express NCA. Fairly homogeneous expression of CEA and NCA was seen in the majority of both breast and lung tumors. Our results indicate that CEA may be an important target for immunotherapy in a large number of patients with breast and lung tumors.     

Monkey antisera with increased specificity to carcino-embryonic antigen (CEA).

Three Macaca irus monkeys were immunized with purified human CEA. One received unmodified CEA and two were immunized with haptenated (4-hydroxy-5-iodo-3-nitrophenyl acetyl) CEA. All three monkeys formed precipitating antibodies to CEA. The antisera did not precipitate the CEA-related normal glycoprotein antigen (NCA). In contrast, CEA-immunized rabbits, sheep and goats invariably form antibodies against NCA. Radio-immunoassays showed the monkey antisera to contain trace amounts of antibodies to NCA but the titers were about 10-1,000 times lower than those in rabbit and sheep sera with comparable anti-CEA titers. These results show that monkeys produce antibodies against CEA, but respond weakly to a normal, CEA-related antigen. The weak response to NCA suggests that monkeys may possess an NCA-related antigen. Antisera lacking reactivity to normal CEA-related antigens may be helpful in establishing a more specific diagnostic test for CEA.     

Decreased expression of biliary glycoprotein in hepatocellular carcinomas

Biliary glycoprotein (BGP) is an adhesion and anti-cell-growth molecule of the carcinoembryonic antigen family. We have earlier demonstrated that BGP mRNA is expressed in hepatocellular carcinomas (HCCs) and the adjacent non-cancerous regions, neither of which express CEA and NCA mRNA. To define an expression level and pattern of BGP at the protein level in HCCs, TS135, a monoclonal antibody (MAb) against BGP, was prepared. This MAb clearly reacted with BGP with a molecular weight of 110 kDa and 85 kDa (BGP-110/85). It cross-reacted weakly with NCA-90 from NCA transfectants, but not at all with CEA-200 from the serum of a colon-cancer patient. The BGP transfectants of cultured hepatocellular carcinoma cHc-4 cells showed Ca²⁻-dependent cell aggregation, which was partially inhibited by modulating BGP on the cell surface with MAb TS135. Immunostaining of non-cancerous liver tissues with MAb TS135 indicated that BGP could be expressed in the bile canalicular domain of hepatocytes. In HCCs, the expression of BGP was predominantly found in the well-differentiated type, where the bile canaliculi and the apical portion of pseudoglands were positively stained, although their staining intensity and stained area were lower and more limited, respectively, than those of non-cancerous regions. The percentage of faintly positive and negative cases (n = 22) from the total (n = 30) was 73%. This suggests that the expression level of BGP decreased in HCCs as compared with adjacent non-cancerous regions. Int. J. Cancer 74:15–19. © 1997 Wiley-Liss, Inc.     

Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD

During the selection of monoclonal antibodies (MAb) raised against purified carcinoembryonic antigen (CEA), two MAbs were identified which immunoprecipitated a glycoprotein of 95 kD present both in perchloric acid extracts of normal lung and on the surface of normal granulocytes. This antigen was distinct from the previously reported normal glycoprotein crossreacting with CEA (NCA) which had a molecular weight of 55 kD. The difference between the smaller and the larger crossreacting antigens termed NCA-55 and NCA-95, respectively, was demonstrated by SDS-polyacrylamide gel electrophoresis, by elution from Sephadex-G200 and by selective binding to a series of anti-CEA MAb. Out of six MAb which all bound CEA purified from colon carcinoma, three did not react with these two crossreacting antigens, one bound only NCA-95, one reacted only with NCA-55 and one reacted with both NCA-55 and NCA-95. Immunoadsorbent purified preparations of 125I labelled NCA-95 and NCA-55 were found useful for the screening of new anti-CEA MAb. In addition, when tested on frozen sections of colon carcinoma, normal spleen, normal lung and pancreas, each type of MAb gave a clearly different pattern of reactivity. The three anti-CEA MAb which did not bind any of the crossreacting antigens stained only the colon carcinoma cells; the MAb binding to either one of the two types of NCA gave a similar pattern of reactivity both on colon carcinoma cells and on granulocytes. However, on normal lung and pancreas, the MAb binding NCA-55 stained granulocytes as well as bronchiolar and alveolar epithelial cells in lung and inter- and intra-lobular duct epithelial cells in pancreas, whereas the MAb binding only NCA-95 stained only the granulocytes. Thus, the newly identified NCA-95 appears to differ from NCA-55 not only in terms of molecular size and antigenicity but also by the fact that in normal lung and pancreas it is found in granulocytes but not in epithelial cells.     

Immunochemical differences among carcinoembryonic antigen in tumor tissues and related antigens in meconium and adult feces

We have isolated carcinoembryonic antigen (CEA)-related antigens from meconium and compared them with those in adult feces. Two CEA-related antigens were detected in meconium [nonspecific cross-reacting antigen 2 (NCA-2) and meconium nonspecific cross-reacting antigen] while four CEA-related antigens were found in adult feces [normal fecal antigen 1, normal fecal antigen 2 (NFA-2), normal fecal cross-reacting antigen, and fecal nonspecific cross-reacting antigen, respectively]. By conventional anti-CEA antisera, NCA-2 in meconium, NFA-2 in adult feces, and CEA in tumor tissues were indistinguishable from each other, but they could be distinguished by specific antibody preparations against a determinant unique to CEA (CEA-distinctive determinant) or to NFA-2 (NFA-2-distinctive determinant). Neither the CEA-distinctive determinant nor the NFA-2-distinctive determinant was detected on the NCA-2 molecule. No antigenic determinants unique to NCA-2 have been detected with the anti-NCA-2 antisera which we have prepared thus far. The molecular weight of purified NCA-2 was estimated to be 150,000 to 170,000 as compared to 160,000 to 170,000 for NFA-2 and 170,000 to 180,000 for CEA. NCA-2 had amino acid and carbohydrate compositions similar to those of CEA and NFA-2. All NFA-2 preparations and about one-half of the CEA preparations were sensitive to Pronase E digestion, which released two antigen fragments from these molecules, but NCA-2-preparations were resistant to such digestion.     

Studies on circulating antibody against carcinoembryonic antigen (CEA) and CEA-like antigen in cancer patients

Characteristics of auto-antibodies for carcinoembryonic antigen (CEA) detected in sera from 3 cancer patients (2 colorectal and 1 breast cancer) were examined. The antibodies belonged to polyclonal immunoglobulin G (IgG). The binding of auto-antibodies with the labeled CEA was inhibited by not only the unlabeled CEA but also NCA-2 (feces and meconium). However, no binding with NCA was observed. Among these auto-antibodies the antibody directed against blood group Lewis determinants which are known to be present in many purified CEA preparations was not found. Previously we had suggested that CEA, NCA-2 and NCA may contain immune determinant in common with alpha 1-acid glycoprotein (AG). These auto-antibodies showed significantly enhanced reactivity for the labeled CEA preparation after purification by anti-AG affinity chromatography in spite of no immunological reaction with AG. These results suggest that auto-antibodies are raised against the common antigenic determinants of both CEA and NCA-2 which do not exist in NCA. These antibodies might be directed to common amino acid sequence shared by CEA and NCA-2, though not excluding the carbohydrate moiety. We surveyed about 500,000 cancer patients but could find only 3 patients who showed a difference in the values of CEA by the indirect and direct method. Thus, the existence of this type auto-antibody to CEA in cancer patients is a rare phenomenon.     

Evolution of the carcinoembryonic antigen family. structures of CGM9, CGM11 and pregnancy-specific glycoprotein promoters.

Earlier studies have demonstrated that the genes of the human carcinoembryonic antigen (CEA) family can be divided into three subgroups, the CEA subgroup (n = 12), the pregnancy-specific glycoprotein (PSG) subgroup (n = 11), and a third subgroup (n = 6). To further characterize the CEA gene family, we have determined the genomic structures of CGM9 and CGM11, analyzed the promoter regions of all eleven PSGs, studied the CGM15-PSG13 intergenic region and the evolutionary relationships beween the CEA family genes. CGM9, a typical CEA subgroup member, was a pseudogene with the exon structure [5'UTR-L-L/N-TM-Cyt-3'UTR]. CGM11 contained a mixture of exons derived from CEA and PSG subgroup genes. The formula of the CGM11 pseudogene was [5'UTR-L-L/N-C-3'UTR]. Thus both genes lacked the IgC2-like domains typically found in CEA subfamily members. The upstream promoter regions of all eleven PSGs were characterized. All PSG promoters lacked the classical TATA and CCAAT elements, but had putative PEA3 box(es), CACCC box(es), a RARE box, and poly (dG-dT) repeats of different lengths. Five PSGs also had an SP1 site. The complete 10-kb intergenic region between CGM15 and PSG13 was sequenced. Clusters of different types of repetitive sequences were seen. The time of divergence of the CEA and PSG subfamilies was estimated to be 107.7 +/- 17.1 million years, or at about the time of human-rodent divergence. Models for the evolution of CEA and PSG and the third family subgroup genes are proposed.     

Characterization of carcinoembryonic antigen-specific monoclonal antibodies and specific carcinoembryonic antigen assay in sera of patients

Six murine monoclonal antibodies (mAbs) reactive with carcinoembryonic antigen (CEA) were prepared. Four of these, CA204, CA205, CA206 and CA208, were specific to purified CEA whereas the other two, NA201 and NA203, were also reactive with the non-specific cross-reacting antigen (NCA). These mAbs were all IgG1, except one IgG2a (CA206), with high affinities for CEA. NA203, CA204 and CA208 appear to define three different epitopes on the CEA molecule as determined by competitive binding assay. These mAbs also reacted with CEA-producing cells. The treatment of the cells with tunicamycin did not affect the binding of the mAbs to CEA-producing cells. None of the above mAbs bound to CEA-related antigens, NCA-2, alpha 1-acid glycoprotein, or blood group antigens. The combination of CA208 (solid phase) and CA204 (tracer) was used to construct a sensitive CEA-specific sandwich ELISA to detect CEA in the sera of patients with various malignant and non-malignant diseases. Particularly when CEA values were low in sera from non-cancerous patients, the above mAbs sandwich assay showed reduced background reactivity with NCA-like substances and permitted the detection of CEA at a level as low as 1 ng/ml.     

Immunological reactivity of mucinous and serous ovarian adenocarcinomas

Comparison of immunological reactivity of glycoprotein antigens extracted from individual cases of mucinous and serous ovarian adenocarcinomas was performed taking into account the immunological relationship with carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein. In all immunological tests, the specific immune sera against perchloric acid extracts of ovarian mucinous and serous cystadenocarcinomas and antisera against the reference antigens mentioned above were used. It was established that: 1) ovarian mucinous and serous adenocarcinomas are immunologically different and possess various tumor-associated antigens, 2) ovarian mucinous adenocarcinomas contain considerable amounts of CEA and NCA, whereas serous type neoplasms show negligible amounts or lack of these antigens; and 3) in both types of tumors, alpha-1-antichymotrypsin and alpha-1-acid glycoprotein activities are found. Immunological data indicate that ovarian mucinous and serous adenocarcinomas derive from separate lineages of epithelium.     

Nonspecific crossreacting antigen (NCA) is a major member of the carcinoembryonic antigen (CEA)-related gene family expressed in lung cancer.

Carcinoembryonic antigen (CEA) is one of the most important tumour markers in the management of human carcinoma, including lung cancer. So far, however, because of the nonspecificity of anti-CEA antibodies, it remains unclear whether the experimental measurements of CEA expression really reflect genuine CEA. In normal lung, nonspecific cross reacting antigen (NCA) has been described as a major component of CEA-related antigens. Recently isolated CEA and NCA cDNA clones enabled us to analyse CEA and NCA expression of in vivo tumour specimens and tumour cell lines at mRNA levels. NCA-specific mRNA (but not CEA-specific mRNA) was detected in all normal lung tissues examined. Of 21 lung cancer tissue specimens, nine expressed both NCA and CEA and five expressed only NCA. Of 16 tumour cell lines, two expressed only NCA and one expressed both NCA and CEA, although its level of CEA mRNA was weaker than that of NCA mRNA. Therefore, CEA-related mRNA expression was always accompanied by NCA mRNA expression; there were no cases of CEA mRNA expression alone. These findings suggest that NCA is a major member of the CEA-related gene family expressed in lung cancer.     

Tumor-associated antigens in female genital tract cancers

The immunologic reactivity of glycoprotein antigens extractable from individual, histologically different ovarian and uterine cancers was studied taking into account their relationship with carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), alpha-feto-protein (AFP), and alpha-1-antichymotrypsin. All studies were performed using specific immune sera against perchloric acid (PCA) extracts of ovarian mucinous cystadenocarcinoma (anti-PCA-CaOm) and cervical squamous cell carcinoma (anti-PCA-CaCx), and antisera against the reference antigens mentioned above. A considerable antigenic heterogeneity and the existence of several immunologically related antigenic systems were found: 1) CEA-like antigens; 2) NCA-type antigens; 3) an antigen different from CEA and NCA present in ovarian mucinous adenocarcinomas and often cross-reacting, but not identical with respective antigens of uterine body and cervical carcinomas; 4) an antigen reacting with anti-alpha-1-anti-chymotrypsin serum; and 5) an antigen reacting with anti-AFP serum.     

CEA-specificity of CEA-reactive monoclonal antibodies. Immunochemical and immunocytochemical studies

Four carcinoembryonic antigen (CEA) reactive monoclonal antibodies Parlam 1, 4, 5 and 6 were studied in respect to reactivity with CEA and its cross-reacting antigens (NCA-1, NCA-2, BGP). In immunochemical studies (ELISA and SDS-PAGE followed by immunoblotting) Parlam 4 did not react with these crossreacting antigens, whereas Parlam 1, 5, and 6 demonstrated variable reactivity with these antigens. As expected, by immunocytochemistry Parlam 1, 5, and 6 stained bile canaliculi in the liver (due to crossreactivity with BGP), pneumocytes and splenic tissue (NCA-1) to an extent comparable with the results in biochemical tests. In contrast with the immunochemical observations, however, Parlam 4 showed slight but distinct reactivity with splenic granulocytes (NCA-1) and hepatic bile canaliculi (BGP). Relative epitope specificity of the monoclonal antibodies was tested in blocking experiments. These showed that Parlam 1 and 5 detect the same epitope on CEA as they block each others binding completely. In other combinations monoclonal antibodies block each others binding only partially, indicating that they detect different or partially overlapping epitopes. These results suggest that CEA specific epitopes may partly overlap with epitopes on crossreacting antigens. In this context, we propose that on crossreacting antigens epitopes exist that are structurally similar to epitopes on CEA or that some epitopes on CEA may consist of a spatial configuration which involves CEA specific as well as non-CEA specific structures. The antibody will show the highest affinity towards CEA, as this molecule contains the uniquely matching or complete epitope and lower affinity towards the crossreacting antigen with an imperfectly matching or only partially available epitope.     

Carcinoembryonic antigen-like substances of human bile. Isolation and partial characterization.

Three species of carcinoembryonic antigen (CEA)-like macromolecules, called the biliary glycoprotein I, II and III (BGP I, II and III) were identified in human bile. BGP I was found in normal gall-bladder bile and hepatic bile but not in bile subjected to inflammation ("white bile"). It was immunologically related to CEA and NGP (the "normal CEA-like glycoprotein". Synonyms: NCA, CCEA-2, etc.). BGP I differed immunologically from CEA in that it lacked the tumor-associated determinants of CEA. It was different from NGP (and CEA) in that it contained BGP I specific determinants as revealed by anti-BGP I antibodies. In bile from gallbladders with obstructed outlet and subjected to cholecystitis, a non-malignant inflammatory process, BGP I was replaced by BGP II and BGP III. Immunologically and physicochemically, BGP II and BGP III appeared to be closely similar to NGP and CEA, respectively.     

Expression of carcinoembryonic antigen and related genes in lung and gastrointestinal cancers.

Carcinoembryonic antigen (CEA), a tumor marker for lung cancers of small cell (SCLC) and non-small cell (NSCLC) types, belongs in a multigene family which includes non-specific cross-reacting antigen (NCA) and biliary glycoprotein 1 (BGP). We used specific cDNA probes and a CEA immunoassay to determine the pattern of expression in normal and malignant lung and gastrointestinal (GI) tissues. Normal lung contained high amounts of NCA and a low concentration of CEA. All 3 genes were expressed discordantly in lung tumors and cell lines. In contrast, all three genes were expressed in most G1 tumor cell lines. In both lung and colorectal cell lines expression of NCA RNA was relatively high, while BGP RNA was relatively low, and the median concentrations of CEA were greater than in corresponding non-malignant tissues. While CEA protein concentrations in lung cell lines were similar to those present in G1 cell lines, the ratio of NCA:CEA RNA was significantly higher in lung cancer lines than in colorectal lines. Thus, NCA constitutes most of the "CEA-like" immunoreactivity previously described in lung cancers. There was excellent concordance between expression of CEA RNA and CEA protein, as well as between concentrations of CEA protein in cell line pellets and supernatant fluids. Of interest, significantly higher rates of CEA expression were present in lung cancers expressing neuroendocrine (NE) markers. The association between CEA expression and NE cell properties is intriguing and may prove to be of clinical interest.     

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.     

Heterogeneity of circulating carcinoembryonic antigen analyzed by sandwich-enzyme immunoassays with different specificities

Nine monoclonal antibodies against carcinoembryonic antigen (CEA) were prepared and used to investigate the immunological and physicochemical heterogeneity of circulating CEA. Two of these, M221-73 and M272-11, recognized "CEA-distinctive" epitopes and they gave sandwich-enzyme immunoassays far less reactive with nonspecific cross-reacting antigen (NCA) and nonspecific cross-reacting antigen-2 (NCA-2). Sandwich-enzyme immunoassays selective for NCA-2 and NCA were also established using suitable monoclonal antibodies as competitive inhibitors against enzyme-labeled antibodies. The studies on the serum CEA levels determined by the "CEA-specific" assay indicated that CEA molecules recognized by M221-73 and M272-11 were generally found in the sera of both cancer patients and normal adults. CEA and related substances in sera were further analyzed by the sandwich-enzyme immunoassays after adsorption on M272-11-coupled immunosorbents and by gel filtration on an Ultrogel AcA-34 column. These studies revealed the presence of a CEA variant detected by the "NCA-2-selective" assay. The variant seemed to be closely related to NCA-2 because it lacked CEA-distinctive epitopes and had an apparent molecular weight similar to that of NCA-2. This variant appeared in the sera of some cancer patients and normal adults.