Monoclonal-antibody-defined human lung tumour cell-surface antigens

A panel of 3 monoclonal antibodies (MAbs) directed against human lung tumour cell-surface antigens has been produced following immunizations with the established small-cell lung cancer (SCLC) cell line, NCI-H69, and with another SCLC cell line, COR-L32, recently derived from clinical material. One MAb, B10/12, reacted strongly with SCLC, immunoprecipitated a protein having an MW of 100kd and failed to react significantly with non-small-cell lung cancer (NSCLC) in radioimmunoassay and in an immunohistochemical assay. MAbs E10/5 and 2G3 reacted extensively with SCLC but also showed significant reactivity with NSCLC. MAb E10/5 immunoprecipitated a protein with an MW of 80kd but no appreciable protein was specifically precipitated by MAb 2G3. Unlike MAb 2G3, both MAbs B10/12 and E10/5 reacted strongly with selected neuroblastomas whereas only MAbs 2G3 and E10/5 reacted significantly with melanoma. All 3 MAbs reacted with breast carcinomas. Other non-pulmonary tumours thus far examined failed to react with the MAbs in radioimmunoassay or immunohistochemical assay. Immunocytochemistry and the use of viable cells in radioimmunoassay confirmed that the antigenic determinants recognized by these MAbs were surface located.     

Cell-surface antigens of human lung tumors detected by mouse monoclonal antibodies: definition of blood-group- and non-blood-group-related antigenic systems

The pattern of antigen expression in human non-small-cell cancers of various histological subtypes has been studied. Mouse monoclonal antibodies (MAbs) were generated following immunizations with cell lines of squamous, adeno- and anaplastic large-cell carcinomas of the lung. Seven non-blood-group-related antigens were defined in addition to 5 antigens related to blood-group determinants. Detailed specificity was established with a large panel of cultured cell lines and normal and neoplastic tissues. MAb F-18 reacted in direct tests with the immunizing squamous lung carcinoma cell line, with 5 out of 5 choriocarcinoma cell lines, but with no other cell lines. No expression of F-18 antigen was observed in any normal or malignant tissue examined. The other 6 non-blood-group-reactive MAbs (F-7, F-8, F-11, F-15, F-16 and F-17) could be distinguished by their reactivity on a panel of cultured cells and tissues. One MAb in this group (F-17) reacted strongly with 19/35 lung tumor cell lines, 32/76 other tumor-derived cell lines, cultured normal kidney cells and fetal lung fibroblasts. This antibody did not react with any normal adult tissues examined, but did react with several cancer tissues including 1/17 lung tumors, 2/4 ovarian cancers and 1/5 colon tumors. Immunoprecipitation tests revealed that 5 of the antigens were glycoproteins: F-18 (Mr greater than 200,000), F-15 (Mr 44,000), F-16 (Mr 90,000), F-17 (Mr 95,000) and F-8 (Mr 95,000). Four MAbs detected Y blood-group antigen (Le(y)), only 2 of which were able to agglutinate O erythrocytes. Another antibody detected X blood-group antigen (Le(x)).     

Representation of epitopes on colon-specific antigen-p defined by monoclonal antibodies

Colon-specific antigen-p (CSAp) is a large molecular-sized protein restricted to normal and neoplastic gastrointestinal tissues and to some mucinous ovarian tumors. Murine monoclonal antibodies (MAbs) were raised against CSAp that was affinity purified with goat polyclonal antibodies from GW-39 human colonic carcinoma xenografts or against the CSAp-producing colon cancer cell line SW-948. Two of the MAbs, designated Mu-2 and Mu-4, recognized a CSAp determinant containing sialic acid, and this epitope was also expressed on bovine submaxillary mucin (BSM). Blocking experiments demonstrated that the Mu-2 and Mu-4 MAbs recognized different determinants. A third MAb, Mu-3, did not cross-react with BSM, but unlike Mu-2 and Mu-4, it did react with human saliva. Reactivity of Mu-3 with saliva did not correlate with major blood group and Lewis-related secretory blood group substances in saliva. This reactivity was not related to sialylated Lewis activity. The fourth antibody, Mu-1, appeared to react with a conformational determinant since its epitope was destroyed by heat treatment or thiol reduction. Enzyme immunoassays have demonstrated that all four epitopes may be expressed on one molecular species.     

Analysis of glycoproteins in cancers and normal tissues reactive with monoclonal antibodies B3 and B1

In this study we show by immunoblotting that B1 and B3, two newly isolated monoclonal antibodies, react with a variety of glycoproteins with different molecular weights expressed in stomach, pancreas, colorectal and breast cancers. The pattern of reactivity differed among cancers arising in different tissues, although no correlation has been observed with the histopathological characteristics of the lesion analysed. MAb B3 and MAb B1, have a limited reactivity with peritumoral tissues, whereas react very strongly with metastatic lesion. Because of the limited reactivity of these antibodies with normal tissue, MAbs B3 and B1, armed with toxin in the form of recombinant immunotoxins, can be useful in treating certain kinds of cancer such as metastatic lesions. However, until current clinical trials are completed, we will not know if they will be helpful in cancer treatment.     

Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor

Background: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. Methods: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. Results: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. Conclusion: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.     

Characterization of a novel neuroglandular antigen (NGA) expressed on abnormal human melanocytes

Murine monoclonal antibodies (MAbs) were produced with reactivity to human malignant melanoma. Six MAbs, 3 of the IgGI (LS113, LS140, LS152) and 3 of the IgG2a (LS59, LS62, LS76) subclasses, were selected for their binding, with an identical pattern of reactivity, to a novel melanoma-associated antigen. As characterized by the enzyme-linked immunosorbent assay (ELISA), these MAbs were found to be positive on n-octyl-beta-D-glucopyranoside extracts of all 10 melanoma cell lines tested and on extracts of 22 metastatic melanoma tumors. The antibodies had minimal reaction with a panel of 14 normal adult tissue extracts. A degree of cross-reactivity was observed with 50% of 39 non-melanoma tumor extracts. The results obtained with the ELISA on cell line and tissue extracts were duplicated using the ABC method of peroxidase staining. The pattern of cross-reactivity, as demonstrated by the intense staining of paraffin-embedded and frozen tissue sections of normal, benign and malignant tissues, defines the recognized protein as a neuroglandular antigen (NGA). Immunoadsorbents made with the antibodies were used to purify the antigen shed from cultured melanomas. All 6 MAbs recognized this purified antigen while 5 other antimelanoma antibodies did not react with it. On gel electrophoresis this antigen is a highly glycosylated glycoprotein with a protein core of 21 kDa.     

The EORTC Melanoma Group exchange program: evaluation of a multicenter monoclonal antibody study

In the framework of the European Organisation for Research and Treatment of Cancer (EORTC), the Immunology and Pathology Subgroups of the Malignant Melanoma Cooperative Group undertook a large multicenter monoclonal antibody (MAb) study. Fourteen laboratories from 7 European countries tested a panel of 23 MAbs for immunohistological staining reactivity for malignant and non-malignant lesions involving the melanocytic lineage. A standardized immunoperoxidase procedure was used and the results were evaluated using a standard protocol and data evaluation form developed in collaboration with the EORTC Data Center. According to this analysis, the antibodies in the panel could be classified into 3 main groups. The first group of MAbs includes those antibodies which stained the majority (greater than 80%) of all primary tumors, irrespective of their Breslow thickness and the majority of metastatic lesions. In addition, these MAbs stained a high percentage of cells within a given lesion. Several antibodies of Group I were likewise reactive with the majority of naevoblasts and with normal melanocytes. The second group of MAbs included antibodies reacting only with a limited number of primary melanomas and metastatic lesions. Antibodies of Group II reacted only weakly, if at all, with normal melanocytes or naevocytes. The percentage of cells within a malignant lesion stained by these MAbs was always rather low. The MAb group III detected surface structures whose expression appeared to be related to tumor progression; they did not react or reacted only weakly with naevi, and they all reacted with a small number of early primary melanomas (less than 0.75 mm). The number of lesions stained increased with increasing Breslow thickness. Our study suggests that the application of a panel of well defined MAbs might be of diagnostic and prognostic value in evaluating malignant melanoma.     

Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonic antigen or HCT-8R cells

A library of 18 monoclonal antibodies (MAbs) reactive with purified carcinoembryonic antigen (CEA) has been prepared. The specificity of these MAbs was tested and they have been separated into nine subgroups, each recognizing a different region of the CEA molecule. Seven MAbs from four of the groups also react with the nonspecific cross-reacting antigen. Some of the MAbs are directed against conformational determinants: three of the MAb groups bind poorly to sodium dodecyl sulfate-treated CEA, while five of the groups are not reactive with reduced and alkylated CEA. Three of the groups react with purified CEA but not with the cell surface CEA of HCT-8R cells, while the other groups react with both forms. The MAbs were tested for binding to fragments of CEA obtained by chemical cleavage and the groups of MAbs were found to react with different subsets of such fragments.     

Private idiotypes located on light and heavy chains of human myeloma proteins characterized by monoclonal antibodies

Idiotypic determinant, an epitope located on the variable region of the heavy or light chain of an immunoglobulin molecule, could be classified into private and public forms. The private idiotype is a marker unique to a single clone of B cell and hence a fingerprint of an individual clone. It could therefore be exploited to monitor expansion of normal or malignant B cells and to target clonally expanded tumorous B cells specifically. In the present study, five murine monoclonal anti-idiotypic antibodies were generated against two human immunoglobulin G (IgG) myeloma proteins. These monoclonal antibodies (MAbs) are produced by hybridoma clones obtained by the fusion of myeloma cells with splenocytes from BALB/c mice immunized with either human IgG1 (three clones) or IgG2 (two clones) myeloma proteins. All MAbs reacted only with the immunizing antigens and had no reactivity with a panel of purified myeloma proteins of four IgG subclasses with different light chains, including IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 4) and IgG4 (n = 5). They reacted with the Fab, but not the Fc fraction of the immunizing antigen and displayed no reactivity with normal human serum or polyclonal IgG. Immunoblotting analysis demonstrated that two of the MAbs react with linear idiotypes on light chain, whereas the remaining three MAbs recognize heavy chain associated idiotopes, either conformational (n = 2) or linear (n = 1). Such MAbs with specificity for private idiotypes could have potential implications for monitoring and specific immunotherapy of B cell malignancies. They also are useful tools to study structural correlates of idiotypes.     

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits

We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.     

Applicability of carcinoembryonic antigen-specific monoclonal antibodies to radioimmunoguided surgery for human colorectal carcinoma

Two carcinoembryonic antigen (CEA)-specific monoclonal antibodies (MAbs), PR1A3 and T84.66, were tested to determine whether they could accurately localize colorectal carcinoma and therefore be applicable in radioimmunoguided surgery (RIGS). Twenty-one tumors by three human colorectal carcinoma cell lines with various levels of CEA expression (KM-12c, C75, and Clone A) were successfully implanted in the intra-abdominal organs of 15 nude mice. The tumors was localized using a portable radioisotope detector (Neoprobe 1000) 48 h after injection of radiolabeled MAbs (10 mCi/mouse) when the precordial counts were <20 per 2 s. Histopathological identification of radiolabeled MAbs were also performed using immunohistochemistry and microautoradiography. Radioactivity counted on a portable radioisotope detector correlated well with that on a gamma counter. The distribution in the blood was significantly greater than in other organs (P < 0.001). Localization indices of the tumor in various organs was from 1.1 to 8.5 in the PR1A3-pretreated mice and 3.0 to 8.6 in the T84.66-pretreated mice. Silver grains and immune staining were distributed in the tumor cells of the PR1A3-pretreated mice, whereas they were in the necrotic debris as well as the tumor cells of the T84.66-pretreated mice. There were significantly more silver grains in the liver in the T84.66-pretreated mice than in the PR1A3-pretreated mice (P = 0.004). The sensitivity and specificity of tumor localization by RIGS were 71.4 and 91.4% in the PR1A3-pretreated mice, whereas they were 60 and 76% in the T84.66-pretreated mice. A study using specific anti-CEA MAbs suggested PR1A3 as an efficient immune probe for RIGS in colorectal carcinoma with a low rate of false-positive detection.     

Oncofetal antigen in Xiphophorus detected by monoclonal antibodies directed against melanoma-associated antigens

Monoclonal antibodies (MAbs) directed against Xiphophorus melanoma cells were developed and tested by indirect immunofluorescence and immunoperoxidase staining for reactivity with a panel of 15 allogeneic tissues and 12 allogeneic cell lines. The reactivity of such MAbs was restricted to melanoma cells from tumor biopsies and melanoma-derived cell lines. In addition, all embryonic cells of all histiotypes from developmental stages later than mid-organogenesis and from corresponding short term in vitro cultures reacted with these MAbs. In contrast, normal tissues and organs from adult fish displayed no reactivity, thus implying that the melanoma-associated antigens detected by the MAbs described are oncofetal antigens.     

Rat monoclonal antibodies produced against rat colorectal adenocarcinomas define tumor- and colon-associated, auto-immunogenic antigens.

Monoclonal antibodies (MAbs) were derived from rats immunized against allo- and syngeneic rat colon carcinomas. Screening was performed by immunohistochemistry modified for MAbs of the same species as the tissue used for frozen sections. From 2 fusions, several MAbs were found that bound to syngeneic tumor tissue but showed little or no staining of normal adult tissues. Another group of MAbs demonstrated an intracellular staining of tumor cells and staining of mucus and goblet cells in the distal 2/3 of the normal colon. A third group of MAbs showed staining of both tumor and normal colon tissue. Staining patterns were reproduced with 6 MAbs selected from the first 2 groups after purification and biotinylation. One MAb, 10B12, recognized an antigen expressed in 10/11 colon carcinomas, the lowest parts of colonic crypts, intracellularly in a subpopulation of pancreatic acinar cells and mucus in antrum. It was used for affinity purification of a high-molecular-weight antigen, to which 3 of the other rat MAbs also bound. This antigen was also bound by antibodies to blood group A and isogloboseries carbohydrate determinants. Competition with the labelled 10B12 MAb for binding to the purified antigen was demonstrated in sera of tumor-bearing and immune rats. Thus, this high-molecular-weight glycoprotein expressed both auto-antigenic, tumor-associated and tissue-type-restricted epitopes. The 10B12 MAb homogeneously stained the majority of colorectal carcinomas and the antigenic determinant was expressed on the cell surface of 12/13 tissue-cultured colon-carcinoma clones. Modulation of the cell-surface antigen phenotype by recombinant rat interferon-gamma markedly increased expression of this determinant.     

Characterization of the colorectal carcinoma-associated antigen defined by monoclonal antibody D612

Monoclonal antibody (MAb) D612 recognizes an antigen expressed on the cell surface of normal and malignant gastrointestinal epithelium. It is a murine IgG2a/kappa which has been previously shown to mediate killing of human colon carcinoma cells using human effector cells (which could be enhanced in the presence of interleukin-2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of MAb D612 immunoprecipitates of extracts of L-[35S]methionine-, L-[3H]leucine-, and D-[3H]glucosamine-labeled human colon carcinoma cells showed that the D612 antigen is a Mr 48,000 glycoprotein. Similar estimates of molecular mass were obtained from SDS-PAGE analyses of MAb D612 immuno-precipitates of radioiodinated extracts of surgically resected colon carcinoma and adjacent normal colonic mucosa. D612 antigen was not detectable in immunoprecipitates of supernatant media from radiolabeled cell cultures, suggesting that the antigen is not readily shed from the surface of cultured cells. The D612 antigen was shown to be clearly distinct from previously described gastrointestinal carcinoma-associated glycoproteins: the D612 antigen shows a migration pattern of SDS-PAGE distinct from those of the antigens recognized by MAbs KS1/4 and GA733, and reciprocal immunodepletion analyses of D-[3H]glucosamine-labeled colon carcinoma cells utilizing MAbs D612 and GA733 revealed no cross-reactivity between these antibodies. Similarly, competitive binding studies between MAbs 17-1A and KS1/4 and MAb D612 revealed no similarity between the epitopes recognized by MAb D612 and MAbs 17-1A and KS1/4. MAbs D612 and 17-1A were also titered in immunoperoxidase staining assays on serial frozen sections of normal and malignant colon. MAb D612 showed a higher titer of immunostaining reactivity with both normal and malignant colon than did MAb 17-1A. MAb D612 showed roughly equivalent immunostaining titers against normal and malignant colon; whereas MAb 17-1A showed higher titer of immunostaining reactivity against the normal colon tissue than against the malignant colon. Flow cytometric analysis of phosphatidylinositol-specific phospholipase C-treated colon carcinoma cells revealed no loss of D612 antigen from the cell surface, suggesting that the mechanisms of attachment of the D612 antigen to the cell surface does not involve linkage to a phosphatidylinositol glycan.(ABSTRACT TRUNCATED AT 400 WORDS)     

Generation of monoclonal antibodies reactive with nuclear proteins of human primary breast tumors

Nuclear proteins were extracted from purified nuclei of human primary breast tumors (BrT) and bladder tumors and of human normal breast, kidney and lymphocytes by enzymatic treatment. SDS-Polyacrylamide gel electrophoresis of nuclear proteins from breast tumors showed different bands in the molecular weight zones from 25 to 220 kDa which were absent or present only as traces in normal breast tissue. Murine monoclonal antibodies (MAbs) have been produced using nuclear extracts of human primary breast tumors as immunogens. Approximately 2,000 hybridomas were generated from 5 hybridizations. According to their reactivity to BrT nuclear extracts and mammary carcinoma cell line MCF-7, seven hybridomas were selected and cloned. They were further characterized with histological immunoperoxidase assays of formaldehyde-fixed BrT paraffin tissue sections. MAb 6A3 particularly gave strong nuclear staining with all BrT specimens while MAb 1D8 showed both nuclear and cytoplasmic staining with only some of them. Specimens from mammoplasty did not react with these MAbs. Immunoblotting of BrT nuclear extracts as developed with MAbs 6A3 and 1D8 revealed major protein bands with molecular weight of 120 and 130 kDa. The potential use of these MAb-defined BrT-related nuclear proteins as markers for human breast cancer was suggested.     

Epstein-Barr virus status and tumour cell phenotype in sporadic Burkitt's lymphoma

Burkitt's lymphoma (BL) biopsy cells and derived cell lines can be grouped according to their patterns of reactivity with 6 selected monoclonal antibodies (MAbs) against B cell-associated surface antigens. Group I cells react only with MAbs J5 and 38.13, recognising the common acute lymphoblastic leukaemia antigen and a BL-associated antigen respectively; group II cells react with J5 and 38.13 and with one or more of a set of MAbs (Ki-24, MHM6, AC2, Ki-1) against "lymphoblastoid" antigens; group III cells react only with these anti-"lymphoblastoid" MAbs. Tumour biopsy cells from 17 cases of sporadic BL, 9 positive for the Epstein-Barr (EB) virus genome and 8 negative, have been analysed during the process of cell line establishment in vitro. In early passage the EB virus-negative BL cells showed either a group I phenotype or gave an additional reactivity with MAb Ki-24 which placed them in group II; these phenotypes remained essentially stable with continued growth of the cell lines for up to 50 passages. By contrast the EB virus-positive BL cells were much more susceptible to phenotypic change in vitro. Although such cells displayed a group I or group II phenotype in early passage, many of the lines soon moved into group III whilst retaining the karyotypic markers indicative of their malignant origin. These observations suggest that a resident EB virus genome can drive the in vitro progression of BL cells towards a more "lymphoblastoid" phenotype. This was confirmed in subsequent experiments where virus-negative BL cell lines were converted to EB virus positivity by in vitro infection. Clearly, therefore, phenotypic analysis of long-established lines can lead to false distinctions being drawn between the EB virus-positive and -negative forms of sporadic BL; both may derive from the same sub-population of target B cells in vivo.     

Immunocytochemical localization of progesterone receptors in breast cancer with anti-human receptor monoclonal antibodies

Monoclonal antibodies (MAbs), recently produced against human progesterone receptors (PR), were used for immunocytochemical localization of PR. The specificity of the immunocytochemical assay for PR was demonstrated by incubation with control MAbs, preabsorption of MAbs with highly purified human PR, and by the cell and tissue distribution of the immunostaining reaction. With human breast cancer cell lines, immunoreactivity was confined to cells that contain PR by steroid-binding assay. Moreover, immunostaining was induced by estradiol in estrogen-responsive cells, MCF-7 and ZR-75-1. In a preliminary study with 33 breast carcinomas, a good correspondence was obtained between immunocytochemical staining and PR content assessed by conventional steroid-binding assay. Immunoperoxidase localization was also obtained with other human target tissues. In normal breast and benign breast disease, immunoreactivity was observed with nuclei of ductal epithelial cells and hyperplastic epithelium. In uterus, immunostaining of endometrium was localized to nuclei of stromal and glandular epithelial cells and in myometrium to nuclei of smooth muscle cells. The effect of the progestin agonist, R5020, and antagonist, RU 486, on PR localization was investigated with the PR-rich T47D human breast cancer cell line. In the absence of hormone, immunostaining was exclusively nuclear. This was true under a number of cell culture conditions designed to eliminate endogenous progestins from the culture medium. Exclusive nuclear localization of PR was not due to a failure of the MAbs to recognize unoccupied PR, since each MAb bound equally well in vitro with different receptor forms. These included liganded and unliganded cytosol PR, molybdate stabilized PR, and nuclear-transformed receptors. Nor was failure to detect cytoplasmic staining due to a selective destruction or loss of unoccupied PR from the cytoplasmic compartment as a result of cell fixation. This was assessed by dot blot immunoassay of PR antigen distribution in subcellular fractions of fixed and unfixed cells. Continuous exposure of cells to R5020 resulted in a transient (30-60 min) increase in nuclear staining intensity (without change in cytoplasmic reactivity), followed by a progressive decline in immunoreactivity. By 24 h of R5020 treatment, the vast majority of cells displayed no immunostaining reaction. These immunocytochemical data are consistent with progestins down regulating their own receptors due to a loss in cellular PR content and not to inactivation of receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein epitopes in carcinoembryonic antigen. Report of the ISOBM TD8 workshop.

To characterize antigenic sites in carcinoembryonic antigen (CEA) further and to investigate whether there are differences between colon tumor CEA and meconium CEA (NCA-2) that can be detected by anti-CEA monoclonal antibodies (MAb), 19 new anti-CEA MAb were analyzed with respect to specificity, epitope reactivity and affinity. Their reactivities were compared with 10 anti-CEA MAb with known CEA-domain binding specificity that have previously been classified into five nonoverlapping epitope groups, GOLD 1-5. Cross-inhibition assays with antigen-coated microtiter plates and immunoradiometric assays were performed in almost all combinations of MAbs, using conventionally purified CEA (domain structure: N-A1B1-A2B2-A3B3-C) from liver metastasis of colorectal carcinomas, recombinant CEA, meconium CEA (NCA-2), truncated forms of CEA and NCA (CEACAM6) as the antigens. The affinity of the MAbs for CEA was also determined. The new MAbs were generally of high affinity and suitable for immunoassays. Three new MAbs were assigned to GOLD epitope group 5 (N-domain binding), 3 MAbs to group 4 (A1B1 domain), 1 to group 3 (A3B3 domain), 3 to group 2 (A2B2 domain) and 3 to group 1 (also the A3B3 domain). Three MAbs formed a separate group related to group 4, they were classified as GOLD 4' (A1B1 domain binding). The remaining 3 MAbs appear to represent new subspecificities with some relationship to GOLD groups 1, 2 or 4, respectively. Five MAbs, all belonging to epitope group 1 and 3, reacted strongly with tumor CEA but only weakly or not at all with meconium CEA, demonstrating that the two products of the CEA gene differ from each other, probably due to different posttranslational modifications.     

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery.

Biparatopic CEA, carcinoembryonic antigen (MAb) was newly designed and tested as to whether it enhanced the accuracy of tumor detection by reducing non-specific binding in experimental radioimmunoguided surgery. Biparatopic MAb was prepared by using cross-linking of reduced Fab' fragments from PR1A3 and T84.66. Fifty-nine tumors from 2 human colorectal carcinoma cell lines with high (KM-12c) and low (Clone A) carcinoembryonic antigen (CEA) expression were successfully implanted subcutaneously on the backs of 42 nude mice. Tumors were localized using 125I-labeled MAbs: IgG, F(ab')(2) and Fab' of PR1A3, and biparatopic MAb of PR1A3 and T84.66. Radioactivity counted on a portable radioisotope detector correlated well with that counted on a gamma counter (p < 0.001). Accumulations of radioactivity in control mice without tumorigenesis were the greatest in PR1A3 IgG-pretreated mice and the least in biparatopic MAb-pretreated mice. Tumors of 2 cell lines did not differ in the distribution of radiolabeled MAbs. Localization indices of the tumor in various organs revealed 1.3 to 4.1 in PR1A3 IgG-pretreated mice, 2.4 to 6.6 in fragment MAbs of PR1A3-pretreated mice and 2 to 4.6 in biparatopic MAb-pretreated mice. Silver grains and immune staining were predominantly distributed in tumor cells of all types of MAb-pretreated mice. Sensitivity and specificity of tumor localization by radioimmunoguided surgery (RIGS) were the highest in the biparatopic MAb-pretreated mice (90.9% and 94.5%, respectively) and the least in the PR1A3 IgG-pretreated mice (50% and 72%). The biparatopic MAb using 2 anti-CEA MAbs against different epitopes achieved a great affinity and avidity with accurate localization of colorectal carcinoma in experimental radioimmunoguided surgery.     

Regional heterogeneity and complementation in the expression of the tumor-associated glycoprotein 72 epitopes in colorectal cancer.

We analyzed the immunohistochemical expression of three epitopes of the tumor-associated glycoprotein 72 (TAG-72) in whole cross-sections of primary colorectal carcinomas and in regional lymph node metastases using monoclonal antibodies (MAbs) B72.3, CC-49, and CC-83, which recognize distinct carbohydrate antigenic determinants. B72.3, CC-49, and CC-83 reacted with 13 of 27 (48%), 25 of 27 (92%), and 21 of 27 (77%) carcinomas, respectively. The immunoreactivity with lymph node metastases followed a similar pattern; MAb CC-49 was again the most reactive of the three antibodies, since it labeled 13 of 15 metastatic lesions. Positive reactions of the MAbs with the primary tumors were not always predictive of the immunorecognition of their metastases. Distinct areas within whole cross-sections of TAG-72-positive primary carcinomas demonstrated marked differences in the expression of the three epitopes. CC-49 tended to react with the highest number of areas and with the highest percentages of carcinoma cells within each area. In no instances did B72.3 demonstrate reactivity superior to that of either CC-49 or CC-83. Tumors negative for the CC-49 epitope in any area also did not express the other two TAG-72 epitopes. However, the comparison of the immunostaining obtained with each MAb in TAG-72-positive primary lesions revealed areas where CC-83 was clearly more reactive than CC-49. Moreover, one lymph node metastasis, negative for CC-49, was recognized by CC-83. Thus, the combined use of MAbs CC-49 and CC-83 resulted in additive immunostaining of primary and metastatic colorectal carcinoma cells. The study provides evidence of intratumoral heterogeneity in the glycosylation pattern of the TAG-72 antigen in colorectal cancer and emphasizes the advantages of cocktails of anti-tumor-associated antigen MAbs in the immunodetection of colorectal tumor cells.