A radioimmunometric assay for circulating tumor-associated glycoprotein (TAG-72) using the antigen determinant CA 72-4

Tumor-associated glycoprotein (TAG-72) is an antigen recognized by monoclonal antibody (MAb) B 72.3 which was generated against a membrane enriched fraction of human mammary carcinoma cells. CA 72-4 is a novel antigen determinant on TAG-72 and is a quantitative radioimmunometric assay system utilizing two MAbs (CC-49, B 72.3) which react with circulating TAG-72 expressed by human carcinomas. We have employed the CA 72-4 RIA system to measure the antigen in sera. The optimum condition for this assay was found to be a 4 hour incubation at 37 degrees C for the first reaction and a 20 hour incubation at 4 degrees C for the second reaction. Under these conditions, intra-assay variation of the control sera was C.V. 3.0-5.2% and inter-assay variation was 5.6-8.3%. The mean +2 SD of CA 72-4 concentration in 468 healthy persons was 3.9 U/ml. Therefore, less than 4.0 U/ml was taken as the cut off level for the CA 72-4 serum assay. The largest population in healthy persons was at the range of 1.5-2.0 U/ml. Only 15 of 468 persons (3.2%) demonstrated serum CA 72-4 levels more than 4.0 U/ml. These data thus indicate the efficacy of CA 72-4 RIA system for the serum assay as a novel tumor marker. The clinical evaluation of CA 72-4 in patients with epithelial malignancies is now underway.     

Expression of tumor-associated glycoprotein-72 (TAG-72) antigen in human prostatic adenocarcinomas

Tumor-specific antigens are usually defined by monoclonal antibodies (MAbs) and can play critical roles in the diagnosis and therapy of carcinomas. Despite advances in the understanding of the molecular genetics of human prostate carcinomas, therapeutic approaches require that tumor-specific markers, preferably on the cell surface, should be defined. In this study, we examined the expression of an oncofetal antigen tumor-associated glycoprotein-72 (TAG-72) in prostatic adenocarcinomas with a Gleason grade of six or higher. Using a second generation MAb CC49 against TAG-72, immunoreactivity was detected in 88% (29/33) of the prostatic cancer tissues. Occasionally, the benign epithelium showed a very faint immunostaining but in most of the specimens, no reactivity was detected. Positive staining was present in the cytoplasm and the cell membrane of the malignant cells similar to reports on other cancer tissues. A weaker staining pattern of this antigen was seen in poorly differentiated areas. A significant negative correlation (r = -0.36, p < 0.05) was observed between TAG-72 antigen expression and Gleason grade. The TAG-72 antigen expression in prostatic adenocarcinomas may be used as a target for radioimmunotherapy by the multivalent single chain antibody CC49 constructs recently generated by our group.     

Tumor-associated glycoprotein (TAG-72) detected in adenocarcinomas and benign lesions of the stomach

Murine monoclonal antibody (MAb) B72.3, prepared against a membrane-enriched extract of metastatic carcinoma and reactive with a high-molecular-weight determinant, designated tumor-associated glycoprotein (TAG)-72, was shown to be reactive immunohistochemically with 97% of a variety of primary adenocarcinomas of the stomach (n = 40). All "early" gastric carcinomas were reactive with MAb B72.3, although the average percentage cellular reactivity was lower than in "advanced" carcinomas. TAG-72 antigen was detected in benign lesions (i.e. adenomatous polyps and hyperplastic polyps) with intestinal metaplasia. Dysplastic lesions characterized by cellular atypia, abnormal differentiation, and disorganized mucosal architecture demonstrated higher TAG-72 expression than non-dysplastic epithelia. In contrast, normal gastric mucosa was generally non-reactive with MAb B72.3. Assays using serial sections of normal, benign and malignant gastric tissues with two MAbs (B1.1 and COL-6) directed against distinct epitopes of carcinoembryonic antigen (CEA) demonstrated differential reactivity, confirming that TAG-72 and CEA are distinct, non-coordinately expressed antigens. Our results suggest that TAG-72 antigen may be expressed in malignant and dysplastic epithelial cells, as well as in intestinalized epithelium of the stomach which has been closely related to subsequent carcinoma development. Hence, MAb B72.3 may be a useful immunohistochemical adjunct for detecting early foci of adenocarcinomas and premalignant lesions of the stomach.     

Analysis of a human tumor-associated glycoprotein (TAG-72) identified by monoclonal antibody B72.3

Monoclonal antibody B72.3 binds a high-molecular-weight tumor-associated glycoprotein identified as TAG-72. This study reports the partial purification and characterization of TAG-72 from a xenograft of a human carcinoma cell line, LS-174T, which expresses high levels of this antigen. The tumor homogenate was initially fractionated by Sepharose CL-4B chromatography. The high-molecular-weight TAG-72, found in the exclusion volume, was then subjected to two sequential passages through B72.3 antibody affinity columns. At each step of the procedure, TAG-72 content was quantitated using a competition radioimmunoassay, and the degree of purification was expressed as the ratio of antigen in units to total protein. The three-step procedure produced a purification of TAG-72 with minimal contamination by other proteins as shown by polyacrylamide gel electrophoresis, followed by staining with Coomassie Blue or periodic acid/Schiff reagent. The density of affinity-purified TAG-72, as determined by cesium chloride gradient ultracentrifugation, was found to be 1.45 g/ml. This density determination, together with the high molecular weight of TAG-72, its resistance to Chondroitinase digestion, the presence of blood group-related oligosaccharides, and sensitivity to shearing into lower-molecular-weight forms suggest that TAG-72 is a mucin-like molecule.     

CA 72-4 measurement of tumor-associated glycoprotein 72 (TAG-72) as a serum marker in the management of gastric carcinoma.

The presence of three distinct serum markers of carcinoma, tumor-associated glycoprotein 72 (TAG-72; as measured by the CA 72-4 assay), CA 19-9, and carcinoembryonic antigen (CEA), was evaluated in 194 patients diagnosed with either malignant (n = 94) or benign (n = 100) gastric disease. Of the 94 patients diagnosed with gastric carcinoma, the percentage of patients whose serum samples were positive for TAG-72, CA 19-9, or CEA was 42.6, 31.9, and 20.2%, respectively. Furthermore, fewer false positive samples were observed for TAG-72 than either CA 19-9 or CEA. The analysis of serum TAG-72, CA 19-9, and CEA levels in patients diagnosed with early (stage I and II) versus advanced (stage III and IV) disease revealed a significantly higher level of TAG-72 and CA 19-9 in the serum of patients with advanced stage gastric carcinoma. The serum samples were also analyzed to determine whether any advantage might be gained by simultaneously measuring two or more of the tumor markers. The data clearly indicate that the measurement of TAG-72 with CA 19-9 significantly increased the percentage of gastric carcinoma patients with positive serum levels of either antigen. This advantage was achieved with no significant increase in the number of false positives. Twenty-one patients were followed postsurgically for up to 3 years to determine whether the appearance or reappearance of TAG-72, CA 19-9, or CEA accurately predicted disease recurrence. Positive serum TAG-72 levels correlated with disease recurrence in 7 of 10 patients, compared with 5 and 2 patients for CA 19-9 and CEA, respectively. The findings suggest that serum TAG-72 as measured by the CA 72-4 assay may be a useful marker for late stage gastric carcinoma and its measurement alone or in combination with CA 19-9 may have utility in the clinical management of gastric carcinoma.     

Tumor-associated glycoprotein (TAG-72) expression in ulcerative colitis

Monoclonal antibody (MAb) B72.3 reactive with the high-molecular-weight (Mr greater than 10(6) tumor-associated glycoprotein (TAG)-72 is being increasingly utilized in vivo and in vitro for a variety of purposes in colon cancer patients. Recent evidence has suggested that the TAG-72 antigen expression may be enhanced in inflammatory bowel disease, particularly ulcerative colitis (Thor et al., 1986a: Cancer Res., 46, 3118-3124). We have utilized 117 paraffin-embedded formalin-fixed colonic specimens from 56 ulcerative colitis patients which demonstrate a spectrum of epithelial abnormalities (reactive atypia, dysplasia, and carcinoma) as well as 11 inflammatory controls to evaluate TAG-72 expression. Our selected patient population all had pan-colitis and demonstrated a generally increasing incidence of dysplasia or carcinoma with duration of disease (20% at 0 to 10 years, 50% at 11 to 20 years, 59% at 21 to 30 years, and 100% at more than 31 years). TAG-72 expression was similar in the control and non-dysplastic colonic epithelia, and increased with low- or high-grade dysplasia as well as carcinomatous lesions (mean cellular reactivities 23.7%, 26.5%, 36.7%, 70% and 84.3%, respectively). Epithelium with low-grade dysplasia exhibited a focal perinuclear, superficial crypt staining (when present). High-grade dysplastic epithelium showed pancytoplasmic, pan-cryptic reactivity. Invasive disease showed cytoplasmic as well as extracellular mucin staining. Biopsies from patients with active disease showed significantly more immunoreactive cells for TAG-72 than patients with quiescent disease. For any given biopsy specimen the percentage of cells reactive did not always correlate with the degree of dysplasia. TAG-72 expression in quiescent disease generally increased with duration of disease, in contrast to active disease which showed no correlation between MAb B72.3 staining and duration of disease. The frequent expression of TAG-72 in actively inflamed colonic mucosa (ulcerative colitis and other colitides) may limit the clinical utility of this antigen for detecting colon cancer in ulcerative colitis patients by serological assay or in vivo radiolocalization techniques. The tendency for TAG-72 expression to correlate with disease duration in patients with quiescent disease and to increase with more severe degrees of dysplasia suggests that the expression of this gene product correlates with the dysplasia-to-carcinoma sequence.     

A radioimmunoassay for the detection of a human tumor-associated glycoprotein (TAG-72) using monoclonal antibody B72.3

Monoclonal antibody (MAb) B72.3 was generated against a carcinoma metastasis and has been shown to bind with a high degree of selectivity to a tumor-associated glycoprotein (TAG-72) found in human colon and breast carcinomas, while showing minimal reactivity to any normal adult tissues. Competition radioimmunoassays have been developed for the detection of TAG-72 in tumors and sera from both athymic mice bearing human carcinoma xenografts and patients with colon, breast and other carcinomas. The distribution of TAG-72 in human tumor xenografts was restricted to tumors originating from the LS-174T human colon carcinoma, with no significant reactivity being detected in human tumor xenografts from a different colon carcinoma, a human breast carcinoma, or a human melanoma. The levels of TAG-72 in clinical material obtained from surgery were examined; high levels were found in colon carcinomas and to a lesser extent in breast carcinomas, while no detectable levels were found in normal tissues. Sera from apparently normal patients contained a mean level of 2.2 units per ml of TAG-72. A level of 3 standard deviations above the mean level of TAG-72 found in normals was employed as a cut-off value to indicate a positive test result in subsequent studies. No patient with inflammatory disease or other benign colon disease exhibited elevated levels of TAG-72. Seven out of 20 patients with other carcinomas had elevated serum levels of TAG-72. The serum TAG-72 levels were compared with the serum levels of antigens recognized by the MAbs currently used to screen sera of carcinoma patients (CEA, GICA, and OC125). It was clearly demonstrated that TAG-72 is different from these antigens and can be found in some sera in which no antigen is detected by otherwise available MAb RIAs.     

Levels of circulating tumor-associated glycoprotein (TAG-72) in patients with carcinoma using a novel tumor marker, CA 72-4

CA72-4 is a novel quantitative immunoradiometric assay system utilizing two monoclonal antibodies CC-49 and B72.3, which recognize a tumor-associated glycoprotein (TAG-72). We have utilized the CA72-4 RIA kit to measure serum levels of TAG-72 in 205 patients with carcinoma and 192 patients without carcinoma. The cut-off value (4.0 U/ml) was obtained according to the levels and the distribution of CA72-4 in 468 healthy individuals. The positive rates in 82 patients with gastric cancer, 55 with colorectal cancer, 24 with pancreatico-choledochal cancer, 36 with breast cancer, and 3 with ovarian cancer were 52%, 55%, 46%, 39%, and 67%, respectively. Fifty percent of the sera from 205 patients with carcinoma demonstrated increased levels of CA72-4, whereas only 10% of the sera from 192 patients without evidence of malignancy showed levels more than 4.0 U/ml. The average level of serum CA72-4 in the patients with carcinoma was 38.6 U/ml, much higher than that (2.7 U/ml) in patients without malignancy. The patients with gastrointestinal cancer at advanced stages or at recurrence showed higher levels of serum CA72-4 than the patients with cancer at early stages. These results thus indicate that CA72-4 is clinically useful as a novel tumor marker, especially for monitoring serum levels of TAG-72 in patients with gastrointestinal cancer, breast cancer, ovarian cancer and other epithelial malignancies.     

Serological mapping of the TAG-72 tumor-associated antigen using 19 distinct monoclonal antibodies.

Monoclonal antibody (MAb) B72.3 has been shown to be of potential utility in the management of human carcinoma via its use in (a) the targeting of carcinoma lesions in colorectal and ovarian cancer patients, (b) immunohistochemical analyses of biopsies and effusions, and (c) serum assays to help define the presence of carcinoma. The B72.3-reactive antigen, designated tumor-associated glycoprotein 72 (TAG-72), has been characterized as a high molecular weight glycoprotein with the properties of a mucin. We report here the utilization of MAb B72.3 and 18 second generation MAbs (generated using purified TAG-72 obtained from a colon carcinoma xenograft as immunogen) to construct a serological map of the TAG-72 molecule. The generation and initial characterization of 10 of the second generation MAbs have been described previously; in addition, eight previously unreported MAbs were used. All 19 MAbs produced immune precipitate lines against purified TAG-72 in double immunodiffusion, indicating that each epitope recognized by a single MAb is present at least twice on the TAG-72 molecule. Immunodepletion analyses utilizing 11 of the anti-TAG-72 MAbs indicated that each recognizes the same molecule or population of molecules. Nineteen competition radioimmunoassays were developed and 19 purified competitor immunoglobulins were used in each assay. The patterns of cross-competition indicated the presence of a complex array of tumor-associated epitopes on the TAG-72 molecule. Some of the MAbs recognized epitopes that were structurally or spatially related to one another, but none appeared to recognize identical epitopes. The spectrum of inhibitory reactivities of these MAbs for TAG-72 binding varied from extremely restricted to more broad inhibition. The serological mapping studies reported here provide information as to the range and nature of the epitopes expressed on the TAG-72 molecule, help form the basis for selecting alternative anti-TAG-72 MAbs for use in potential clinical applications, and further define the nature of this oncofetal antigen.     

Purification and composition of the human tumor-associated glycoprotein (TAG-72) defined by monoclonal antibodies CC49 and B72.3

Human mammary and other carcinoma cells secrete and express on their cell surfaces complex, mucin-like glycoproteins (Mr greater than 10(6] that are recognized as tumor-associated antigens by a variety of monoclonal antibodies (MAbs). One such MAb, B72.3, has been extensively studied as to range of reactivity for a variety of carcinomas versus normal tissues. The B72.3-reactive antigen, designated tumor-associated glycoprotein (TAG)-72, which is a high-molecular-weight mucin, was partially purified and used as immunogen to produce second generation anti-TAG-72 MAbs. One of these second generation MAbs, CC49, was chosen to be used to develop a procedure to yield preparative amounts of purified antigen suitable for analyzing amino acid sequence. One of the reasons for the selection of CC49 for these studies is that it demonstrated a higher Ka for TAG-72 compared with B72.3. Xenografts of LS174T cells (a human colon carcinoma cell line) grown in nude mice were solubilized, extracted with several chaotropic agents and treated with perchloric acid. The acid soluble antigen was subjected to MAb CC49 affinity chromatography, gel filtration, and ion-exchange chromatography using fast protein liquid chromatography and high-performance liquid chromatography methodologies. A double-determinant liquid competition radioimmunoassay for TAG-72 showed greater than a 1000-fold purification. Radiolabeled protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an apparently homogeneous high molecular weight mucin and a small amount of an additional protein with an apparent Mr of 63,000. Chemical deglycosylation using trifluoromethanesulfonic acid yielded low molecular weight proteins, which could be analyzed for amino acid sequence, and also became susceptible to tryptic digestion. The amino acid composition of the purified TAG-72 mucin was similar to that of other purified mucins.     

Characterization of the shed form of the human tumor-associated glycoprotein (TAG-72) from serous effusions of patients with different types of carcinomas

Monoclonal antibody (MAb) B72.3 binds a high molecular weight tumor-associated glycoprotein designated TAG-72. This study reports the isolation and characterization of secreted TAG-72 directly from effusions of ovarian, colorectal, pancreatic, and endometrial carcinoma patients and compares them to TAG-72 derived from the LS-174T colon carcinoma xenograft. The B72.3-reactive antigen, TAG-72, was used as immunogen to produce second generation anti-TAG-72 MAbs. One of these second generation MAbs, CC49, had a higher affinity than that of B72.3 and was utilized as an affinity reagent in a procedure to purify the TAG-72 present in the serous effusions of carcinoma patients. A three-step purification procedure, utilizing heat extraction, CC49 antibody affinity chromatography, and gel filtration chromatography, resulted in 1000- to 4400-fold purifications of the TAG-72 derived from effusions, as analyzed using a double-determinant radioimmunoassay. Radiolabeled TAG-72 from each of the effusions demonstrated similar high molecular weight bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Similar results from the various effusions were also obtained in Western blotting analyses. Analyses of TAG-72 from the different effusions in radioimmunoassay using five different anti-TAG-72 MAbs revealed similar binding patterns. The results of these studies thus indicate that TAG-72 obtained directly from patients with ovarian, colorectal, endometrial, and pancreatic carcinomas and the LS-174T xenograft are highly similar in terms of immunochemical properties and antigenic profile.     

Tumor-associated glycoprotein (TAG-72) in ovarian carcinomas defined by monoclonal antibody B72.3

Murine IgG1 monoclonal antibody (MoAb) B72.3, reactive with a high molecular weight glycoprotein complex [designated tumor-associated glycoprotein-72 (TAG-72)] was shown, with the use of the avidin-biotin-complex-immunoperoxidase technique and surgically resected tissues, to be reactive with a variety of histologic tumor types. TAG-72 is expressed in at least 5% (and up to 100%) of the malignant epithelial cells in 77% (n = 52) of human primary cancers and 71% (n = 31) of metastatic ovarian cancers of the common "epithelia" histologic category. Of these, several histologic types, including serous and mucinous cystadenocarcinomas, undifferentiated carcinomas, and less common types of ovarian carcinoma, were all shown to express TAG-72. In contrast, normal ovarian tissues and 26 of 27 benign ovarian tumors of various histologic types failed to express similar levels of TAG-72. Of interest is the 1 benign tumor that demonstrated unusual glandular complexity, as well as 3 tumors designated as borderline malignancy, that contained elevated TAG-72 expression. MoAb B72.3 also was used successfully to detect ovarian carcinoma cells in 28 cytologic preparations of human serous effusions and peritoneal washings. The reactivity of MoAb B72.3 was shown to be distinct from that of MoAb OC125 and an anti-CEA MoAb B1.1. The potential applications of MoAb B72.3 in the study of human ovarian cancer cell populations, as well as in several aspects of the management of human ovarian cancer, are discussed.     

Tumor-associated antigen TAG-72: Correlation of expression in primary and metastatic breast carcinoma lesions

Variability of tumor-associated antigens among and within human tumor cell groups presents a potential problem in the development and optimization of immunodiagnostic and therapeutic procedures for cancer. We determined the degree of expression of a tumor-associated antigen in the primary and metastatic lesions of 23 patients with infiltrating ductal carcinoma; this was accomplished using monoclonal antibody B72.3, an IgG1 generated against membrane-enriched fractions of human metastatic breast carcinomas and reactive with a 220,000-400,000 d glycoprotein complex, termed TAG-72, and the avidin-biotin complex immunoperoxidase method on fixed tissue sections. Sixteen of the 23 breast carcinomas (70%) demonstrated MAb B72.3 reactivity (range 5% to 100% of tumor cells staining). Reactivity of lymph node metastases was present in 14 of 21 patients (67%). MAb reactivity in metastases to distant sites, including bone, adrenals, liver, skin and effusions, was present in 10 of 18 patients (56%). In one patient, neither the primary carcinoma nor the metastasis to the lymph node demonstrated reactivity. There was a statistically significant positive correlation between MAb B72.3 reactivity in both primary and lymph node metastases (Kendall's Correlation Coefficient = 0.60, p = 0.0006) and between lymph node and distant metastases (Kendall's Correlation Coefficient = 0.48, p = 0.02) of the same patient. No correlation existed between antibody reactivity seen in the primary and that found in the distant lesions of that patient. These studies thus demonstrate that monoclonal antibody B72.3 can detect expression of a tumor-associated antigen in both primary and metastatic infiltrating ductal carcinoma lesions, and may prove valuable in the understanding of tumor biology of metastases and as a means for diagnosing occult disease.     

Monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 72) on a novel pancarcinoma antigen (TAG-72)

CA 72 is a monoclonal antibody (MAb) -defined antigenic determinant expressed on a pancarcinoma antigen (TAG-72) found in more than 85% of human colorectal carcinomas. An immunoradiometric assay has been developed using the murine MAb B72.3 to quantitate CA 72 in human serum. In a simultaneous immunoradiometric assay, the mean CA 72 concentration in 1,099 serum samples from healthy blood donors was 1.83 +/- 2.03 (SD) units/ml. If the upper limit of normal was set at 10 mu/mol of serum, a value including 99% of healthy blood donors, only 4 of 101 serum samples (4%) from patients with benign disease were elevated, whereas 15 of 26 (58%) and 14 of 25 (56%) of rectal and colon carcinoma patient sera, respectively, were positive. Serum samples from 84 benign colorectal disease cases were examined; of these, 0 of 28 (0%) colorectal adenoma, 1 of 39 (3%) ulcerative proctocolitis, 0 of 15 (0%) diverticulosis, and 0 of 2 (0%) irritable bowel disease sera contained more than 10 mu/ml CA 72. At a reference value of 20 mu/ml, 0 of 101 (0%) benign disease and 2 of 1,060 (0.2%) blood donor sera had elevated values, whereas 10 of 26 (38%) and 9 of 25 (36%) rectal and colon patient sera, respectively, remained positive. The majority of patients with pancreatic and ovarian cancer, and a significant fraction of stomach cancer patient sera, also contained elevated levels of CA 72. The ability of this assay to discriminate between malignant and benign diseases suggests its further evaluation for monitoring and diagnosis in groups at risk for development of cancer.     

Expression of TAG-72 in normal colon, transitional mucosa, and colon cancer

Monoclonal antibody (MAb) B72.3 detects an epitope carried by high-molecular-weight mucins (tumor-associated glycoprotein, TAG-72) recently identified as sialyl-Tn. B72.3 MAb has a restricted pattern of reactivity with normal tissues but it reacts with a high proportion of epithelial cancers. To determine the possible relationship between neoplastic transformation and reactivity with B72.3 MAb, we have analyzed samples of normal colon, colon cancer and transitional mucosa (mucosa adjacent to colorectal cancer) or reactive mucosa (mucosa adjacent to squamous carcinoma of the anal canal, or mucosa overlying lymphoma). B72.3 MAb reacted strongly with 21/21 specimens of transitional mucosa and with 17/21 specimens of adjacent colon cancer. Reactivity of B72.3 MAb with transitional mucosa was strong and homogeneous, whereas reactivity with cancer tissue was weaker and more heterogeneous. Reactive mucosa adjacent to squamous carcinoma or lymphoma was also reactive with B72.3 MAb. Our findings show that, in the colon, expression of TAG-72 antigen occurs during the process of epithelial cell transformation but is also regulated by factors unrelated to the process of carcinogenesis.     

Comparative studies on the expression of tumor-associated glycoprotein (TAG-72), CA 19-9 and DU-PAN-2 in normal, benign and malignant pancreatic tissue

Expression of tumor-associated glycoprotein (TAG-72) was examined by immunohistochemistry in pancreatic specimens from normal donors, and from patients with chronic pancreatitis and pancreatic cancer, and was compared with expression of CA 19-9 and DU-PAN-2. In the normal pancreas, TAG-72 was expressed in fewer ductal and ductular cells than were CA 19-9 (p less than 0.05) and DU-PAN-2 (p less than 0.01 and 0.001 respectively), whereas in chronic pancreatitis all 3 antigens were expressed in ductal cells but only CA 19-9 and DU-PAN-2 in ductular cells (p less than 0.001). In the specimens from normal pancreas and chronic pancreatitis cases, TAG-72 was localized in the Golgi region, whereas CA 19-9 and DU-PAN-2 showed diffuse cytoplasmic and glycocalyx patterns. In pancreatic cancer, although the rate of expression of all 3 antigens was similar, their cellular localization differed significantly: only TAG-72 was expressed in the Golgi region (p less than 0.001), whereas CA 19-9 showed mainly glycocalyx (p less than 0.05) and/or intra-luminal patterns (p less than 0.01) compared with that of the other 2 antigens. We conclude that, due to differences in expression of TAG-72 in benign versus malignant cells, the monoclonal antibody against TAG-72 (B72.3) may be suitable for radio-immunodetection or radio-immunotherapy of pancreatic cancer.     

Generation and characterization of B72.3 second generation monoclonal antibodies reactive with the tumor-associated glycoprotein 72 antigen

Monoclonal antibody (MAb) B72.3 was generated using a membrane-enriched fraction of a human mammary carcinoma biopsy. It has demonstrated reactivity to the majority of human adenocarcinomas including colorectal, gastric, pancreatic, ovarian, endometrial, mammary, and non-small cell lung cancer and no or weak reactivity to normal adult tissues, with the exception of secretory endometrium. The B72.3-reactive antigen, termed tumor-associated glycoprotein (TAG)-72, has been purified and used as an immunogen to generate B72.3 second generation MAbs. Since the source of purified TAG-72 was a human colon cancer (CC) xenograft, these MAbs have been given a CC designation. Twenty-eight CC MAbs, all immunoglobulin Gs, have been generated and shown to be reactive with TAG-72 and via both radioimmunoassay and immunohistochemical analyses show differential reactivity to carcinoma versus normal adult tissue biopsies. Nine CC MAbs (CC11, 15, 29, 30, 40, 46, 49, 83, and 92) were selected for further characterization. As a result of analyses using direct-binding radioimmunoassay to a range of human carcinomas, Western blotting, live cell surface binding assays, five liquid competition radioimmunoassays, and Ka measurements, all nine CC MAbs could be distinguished from each other and from B72.3. The Ka of B72.3 was determined to be 2.54 X 10(9) M-1; all the CC MAbs demonstrated higher KaS with MAbs CC92, 49, and 83 having KaS of 14.26, 16.18, and 27.72 X 10(9) M-1, respectively. These studies thus demonstrate that one or more of the anti-TAG-72 CC MAbs may be more efficient than B72.3, or useful in combination with B72.3, toward the further study of human carcinoma cell population and the diagnostic and therapeutic procedures presently utilizing MAb B72.3.     

Evidence of specificity of radiolabeled phage display peptides for the TAG-72 antigen

The TAG-72 glycoprotein is highly expressed in many tumor types and has been considered a target for tumor imaging. In this work, we used the f88-4/Cys6 phage library of constrained 16 mer peptides to select those that demonstrate binding to TAG-72. Three consensus peptides were identified: NPGTCKD-KWIECLLNG (clone A); NLIWCRKEFARCTSDM (clone B); and LKNYCRKCSNRCTPTG (clone C). The phage of clones containing these peptides were radiolabeled with technetium-99m (99mTC) at 90% radiochemical purity and were incubated with TAG-72-positive LS174T colon cancer cells. The phage of clones A and B bound significantly higher by 4.5-fold and 1.5-fold than that of a nonspecific control phage. The 99mTc-labeled phage of clones A, B, and control were also administered intravenously to mice with LS-174T tumors. The accumulation of phages from clone A showed a slightly statistically higher accumulation in the tumor (0.51% ID/g), compared to phages of clone B and control phage (0.28% and 0.29% ID/g; p=0.049 for both). In conclusion, the peptide expressed by clone A phage showed evidence of significant specific binding when presented as the radiolabeled phage to tumor in vivo and especially in vitro with cells and solid tumor. The results suggest that this peptide when free of the phage may have potential for the imaging and possibly radiotherapy of TAG-72-expressing cancers.     

肿瘤相关糖蛋白72和葡萄糖调节蛋白94在乳腺良恶性病变组织中的表达及其意义

目的:研究乳腺良恶性病变组织中肿瘤相关糖蛋白72(tumor-associated glycoprotein, TAG72)和葡萄糖调节蛋白94(glucose-regulated protein 94, GRP94)的表达及其临床病理意义.方法:采用免疫组织化学法检测60例乳腺癌组织和30例乳腺良性病变组织中TAG72和GRP94的表达,并进行评分.应用Kaplan-Meier法对乳腺癌患者进行生存分析.结果: 乳腺癌组织中TAG72和GRP94表达的阳性率分别为68.3%和63.3%,其总评分分别为2.53±1.86和2.73±2.23;乳腺良性病变组织的TAG72和GRP94表达阳性率分别为10.0%和13.3%,其总评分分别为0.37±1.13和0.47±1.22;乳腺癌组织与乳腺良性病变组织相比,差异均有统计学意义(P = 0.000).导管内癌、组织学分级为Ⅰ或Ⅱ级、肿瘤最大径≤3 cm、无淋巴结转移、雌激素受体阳性、p53阴性及CA15-3阴性乳腺癌患者的TAG72和GRP94表达阳性率及其评分均明显低于浸润性导管癌(P＜0.05)、组织学分级Ⅲ级(P＜0.01)、肿瘤最大径＞3 cm(P＜0.05)、淋巴结转移(P＜0.01)、雌激素受体阴性(P＜0.05)、p53阳性(P＜0.01)和CA15-3阳性(P＜0.01)的乳腺癌患者.TAG72和GRP94表达阳性的乳腺癌患者及两者均表达阳性的乳腺癌患者的术后生存期明显短于阴性表达者(P＜0.01).COX多因素分析结果显示,TAG74和(或)GRP94表达阳性均与术后生存率呈负相关,与死亡率呈正相关,均是独立的预后因素.结论:TAG72和GRP94表达在乳腺癌的发生、发展、临床生物学行为及预后方面可能具有重要作用,TAG72和(或)GRP94表达阳性的乳腺癌患者预后不良.     

Immunoreactivity and prognostic value of tumor-associated glycoprotein 72 in primary gallbladder carcinoma

AimThe purpose of this study was to investigate the expression of tumor-associated glycoprotein 72 (TAG-72) in primary gallbladder carcinoma (PGC) with an attempt to determine the potential usefulness of it in diagnostic and prognostic applications.MethodsTissue samples from 118 patients with PGC, 30 patients with chronic cholecystitis, and 20 normal gallbladders were stained with anti-TAG-72 antibodies for immunohistochemical analysis. Then, the clinical outcome of the patients after a maximum follow-up of 5 years was determined in 110 out of 118 patients.ResultsClinicopathological characteristics of the carcinomas and clinical outcome of the patients were associated with the TAG-72 expression. TAG-72 was expressed more frequently in cancerous tissues of larger size, with lymph nodes metastasis, and with poor differentiation. Especially, a statistical association was found with more advanced UICC stages of the disease (55.77%, 65.38%, 92.86%, 93.75% and 100% in stages IA, IB, IIA, IIB, and III, respectively, p = 0.02). Using a proportional hazard model, the survival rate of the patients with PGC expressing TAG-72 was significantly lower than the patients without TAG-72 expression (p < 0.01), and including information of TAG-72 staining patterns within cancerous tissues along with clinical cancer staging may improve the accuracy of predicting patients' prognosis.ConclusionThese data suggest that TAG-72 expression is associated with clinicopathological parameters of aggressiveness in PGC. The detection of it combined with cancerous staging may increase the ability of investigators to predict the prognosis of patients with PGC.