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.     

Immunohistochemical localization of carcinoembryonic antigen (CEA), CEA-S, and nonspecific cross-reacting antigen (NCA) in carcinomas of lung

Antisera to carcinoembryonic antigen (CEA), to a physicochemical subset of CEA, namely CEA-S, and to nonspecific cross-reacting antigen (NCA) were used for the immunohistochemical localization of these antigens in human bronchogenic carcinomas using a triple layer immunoperoxidase technique. The study is based on an analysis of tumors from 130 patients. CEA, CEA-S, and NCA were all identified in the membrane and/or cytoplasm of neoplastic cells, and a good correlation between the antigens was observed in a majority of tumors. The presence or absence of these tumor-associated glycoproteins appeared to be correlated with the histologic type of the tumors, especially in small cell anaplastic carcinoma and adenocarcinoma, and the degree of histologic differentiation of adenocarcinomas correlated positively with these tumor-associated antigens. Data from this group of patients suggest that analysis of tissue CEA at the time of biopsy or surgical resection may facilitate a more objective interpretation of serial plasma CEA assays.     

Localisation of monoclonal antibodies reacting with different epitopes on carcinoembryonic antigen (CEA)--implications for targeted therapy.

Antibody targeting has potential for selective delivery of cancer therapy. However, there is a wide variation in the degree of antibody localisation in individual patients with colorectal adenocarcinoma. Colorectal adenocarcinomas are composed of glandular structures separated from fibrovascular stroma by a basal lamina which may represent a significant barrier to extravasated antibody. Basement membrane-associated CEA epitopes may be more accessible to antibodies than those which are cytoplasmic or lumenal. We have investigated by immunohistochemistry and in vivo localisation, the extent to which distribution of antigen epitopes influences targeting. Two monoclonal antibodies (A5B7 and EA77) recognising non-overlapping CEA epitopes were reacted immunohistochemically with samples of 39 tumours. Intensity and site of reaction were assessed for basement membrane, cytoplasmic or lumenal surface association. 125I-labelled antibodies were injected into nude mice bearing LS174T tumour. Per cent injected activity per gram was measured in tumour and normal tissues, 24, 72 and 168 h later. Tissues reacted immunohistochemically for CEA were autoradiographed to assess the relationship of injected antibody to target antigen. Immunohistochemistry showed that A5B7 antibody favours basement membrane aspects of malignant glands; in contrast, EA77 concentrated generally on lumenal surfaces. In vivo localisation showed that per cent inj.act g-1 in tumour for A5B7 reached 36.5% at 24 h. EA77 localised to a lesser extent (9.1% at 24 h), despite a longer circulatory half-life. Autoradiography combined with immunohistochemistry showed A5B7 reacting with antigen close to vasculature after 24 h, slowly penetrating deeper parts of the tumour by 72 h. In contrast, EA77 was confined mainly to fibrovascular stroma, showing little labelling of antigen-positive tumour cells. Localisation differences between A5B7 and EA77 may partly be due to accessibility of epitopes on tumour cells.     

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.     

Ultrastructural localization of carcinoembryonic antigen (CEA) on ultrathin cryosections of human tumours

A new, more direct method for reliable localization of CEA on human tumours has been applied at the ultrastructural level. Freshly prepared biopsy material from breast and gastric tumours, as well as xenografted colon tumours from mice, and a colon tumour cell line (LS 174 T), were briefly fixed in glutaraldehyde and frozen in liquid propane. Ultrathin cryosections were labelled with monospecific CEA antibodies by applying gold-coupled second antibodies. Variations in the labelling patterns were found when comparing breast, colon, and gastric cancers. Apart from breast tumours, all other samples reveal significant extracellular CEA positivity on the cell membrane. Intracellular localization has been found on the endoplasmic reticulum, nuclear membrane, and, in breast tumours, on electron dense bodies. These investigations confirm our previous experimental results using preembedding labelling techniques before Epon inclusion.     

Immunohistochemical evaluation of nonspecific cross reactive antigen and carcinoembryonic antigen (CEA) in urinary bladder carcinoma

The immunohistochemical expression of CEA in formalin-fixed paraffin sections in urinary bladder carcinomas was compared to the use of polyclonal anti-CEA antiserum (P-CEA), NCA-absorbed anti-CEA (NCA-aCEA) and monoclonal antibody to CEA (M-CEA). The urinary bladder carcinomas examined consisted of 19 cases of transitional cell carcinoma (TCC) and 7 cases of squamous cell carcinoma (SCC). Both TCC and SCC were positive for CEA with the use of P-CEA and NCA-aCEA, and the degree of staining was markedly dependent on the grade of malignancy in TCC. However, the reaction to M-CEA was generally very weak or negative in TCC and SCC. In SCC, the staining reaction was confined to keratinized foci and not found in all malignant tumour cells when polyclonal CEA antiserum was used. These findings indicate that positive reactions seen with conventional CEA antibodies (P-CEA and NCA-aCEA) are possibly related to NCA and that urinary bladder carcinoma may contain relatively more NCA than true CEA.     

Immunocytochemical localization of carcinoembryonic antigen (CEA) and transport of CEA into the blood vessels in gastric cancer

CEA was localized in the luminal border, cytoplasm, but not in mucus, in Signet ring cell carcinoma (sig) and mucinous carcinoma (muc). Electronmicroscopically, CEA was localized in the glycocalyx of the microvilli and microvesicles of the cytoplasm. The histologically different cancer types showed no difference in the localization of T-CEA. We also studied P-CEA elevating factors in 38 CEA-positive (++) patients manifesting subserosal (ss) or deeper invasion. No remarkable findings were obtained. When P-CEA elevating factors were studied in 20 patients with (+) CEA reaction and ss or deeper invasion, we found that the incidence of por was high in P-CEA negative cases. In particular, the por incidence was significantly low (p less than 0.01) in patients with scirrhous type. High P-CEA levels were detected in blood adjacent to the cancer. Among 6 cases with negative P-CEA in the vessels adjacent to the cancer whose T-CEA reactions were (++) or (+), and who manifested ss or deeper invasion, there was a high incidence of por (5 cases) and scirrhous type (4 cases), histologically.     

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.     

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…     

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.     

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.     

Immunological study of carcinoembryonic antigen (CEA) and a related glycoprotein

A comparison has been made of the immunological properties of CEA (carcinoembryonic antigen) and another perchloric acid-soluble macromolecule which occurs in colonic and certain other carcinomata and which is here termed CEX. By using a variety of antisera it was shown that the two substances share common antigenic groups as well as having characteristic ones of their own. These latter groups have enabled the preparation of (a) antisera which give a gel diffusion line only with CEA and (b) and antiserum which gives a line only with CEX. No immunological difference could be found between CEX and the NGP of Mach or the NCA of von Kleist and Burtin. CEX was found in foetal gut, in plasma and associated with CEA in virtually all the tissues and fluids in which the latter occurs; the two appear to go hand-in-hand and no proof was found that CEX is either less or more cancer specific than CEA—it is merely found in greater quantity; neither substance showed absolute cancer specificity. The usefulness of a radioimmunoassay for CEX is discussed, and also the possibility of interference by CEX in the radioimmunoassay for CEA. Evidence of two molecular species of CEA has been found.     

Immunohistochemical demonstration of carcinoembryonic antigen (CEA) and its correlation with grading and staging on tissue sections of urinary bladder carcinomas

In 150 transitional cell carcinomas (TCC) of the urinary bladder, 50 each of Grades 1, 2, and 3, the content of carcinoembryonic antigen (CEA) was examined immunocytochemically and correlated to grading and staging. Fifty-seven percent of the TCC contained CEA-positive tumor cells. Their distribution in tumor tissue is described. They were found in 24% of Grade 1 cases, in 72% of Grade 2 carcinomas, and in 76% of Grade 3 tumors. None of the Grade 1 cases contained more than 10% CEA-positive cells, whereas 34% and 40% of the Grade 2 and 3 tumors, respectively, revealed more than 10% CEA-positive tumor cells. According to the correlation found between grading and staging, the percentage of TCC containing CEA-positive tumor cells was 34% in pTA, 59% in pT1, and 80% in pT2/3 tumors. The results show a correlation between CEA content of tumor tissue and histopathologic malignancy in TCC of urinary bladder.     

A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA)

Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.     

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.