Motohisa Kuwahara

Reducing interference from heterophilic antibodies in a two-site immunoassay for carcinoembryonic antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer

To reduce heterophilic antibody interference in a two-site immunoassay for carcinoembryonic antigen (CEA), we utilized a human/mouse chimeric antibody to CEA as the tracer. One mouse monoclonal antibody (MAb), F82-61, which reacts with an epitope present on the domain N of CEA, was immobilized on 96-well polystyrene microtiter plates. A human/mouse chimeric antibody (Ch F11-39), which recognizes an epitope present on the domain B3 of CEA, was biotinylated for the tracer (Ch F11-39 system). Another MAb F11-39, the parental MAb of Ch F11-39, was also biotinylated and used as the control tracer (F11-39 system). For a fair comparison, the same 503 serum samples from healthy individuals were simultaneously assayed in the present study. When a tentative common reference limit of 5 ng/ml was used, the false positive rate with the Ch F11-39 system was only 2.8% (14/503) and that with the F11-39 system was 29.0% (146/503). Adding normal mouse serum (NMS; 1%) or a mixture of purified mouse IgG subclasses (heterophilic blocking reagent (HBR, 15 micrograms/test)) to the F11-39 system reduced the false positive rate from 29.0% to 6.2% (31/503) or 4.8% (24/503), respectively, suggesting that heterophilic antibodies reactive with mouse IgG gave rise to the high positive rate in normal populations with the F11-39 system. On the other hand, the false positive rate with the Ch F11-39 system was only slightly reduced from 2.8% to 2.6% (13/503) or to 2.0% (10/503) by adding NMS or HBR to the Ch F11-39 system. The false positive rates with two commercially available assay systems, CEA Roche EIA.DM or Abbott IMx CEA, were 5.4% (27/503) and 5.8% (29/503), respectively, which both corresponded roughly to that with the F11-39 system including NMS or HBR. These results indicate that the application of human/mouse chimeric antibodies in two-site immunoassays is more effective for reducing interference from heterophilic antibodies than the adding of NMS or purified mouse IgG in the assay using conventional MAbs.

BINDING REACTIVITY OF MONOCLONAL ANTI-CARCINOEMBRYONIC ANTIGEN (CEA) ANTIBODIES WITH CELL MEMBRANE-BOUND CEA AND WITH FREE CEA IN SOLUTION

Binding reactivities of 62 anti-CEA MAbs from 10 different research groups with cell membrane-bound CEA and with free CEA in solution were compared by inhibition of MAb binding to CEA-expressing tumor cells by free CEA. Bindings of 30 MAbs to the cell membrane-bound CEA (280 ng CEA/2 x 10(5) cells) were inhibited by approximately equal amounts of free CEA, indicating that binding affinities of about half the MAbs for cell membrane-bound CEA are similar to those for free CEA, respectively. Bindings of 15 MAbs to the cell membrane-bound CEA were easily inhibited by free CEA of less than half the amount of the cell membrane-bound CEA, while inhibition of bindings of the remaining 17 MAbs required twice more free CEA than the amount of cell membrane-bound CEA, showing that about one-fourth of the MAbs have higher affinities for free CEA and the remaining about one-fourth of the MAbs possess higher affinities for cell membrane-bound CEA. These results help form the basis for selecting the anti-CEA MAbs for use in clinical applications, such as serum CEA assay, tumor imaging and immunotherapy.

High levels of hepatocyte growth factor/scatter factor in diffuse‐type bronchioloalveolar cell carcinoma

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen for various neoplastic cells, including neoplastic bronchial epithelia.

A small randomized phase III single-center trial on postoperative UFT administration in patients with completely resected non-small cell lung cancer.

Our objective was to clarify the efficacy of UFT administration after the complete resection of non-small cell lung cancer (NSCLC) at a single-center institution, avoiding the biases produced by interinstitutional differences. A total of 30 patients who underwent the complete resection of NSCLC at our hospital between 1987 and 2001 were randomly assigned to a control group or to a UFT group (400 mg/day for 2 years). Thirteen patients were assigned to the control group and 17 patients were assigned to the UFT group. The overall survival rate, disease-free survival rate, patient compliance and adverse effect of the UFT treatment were then analyzed. The overall survival and disease-free survival rates of the UFT group were superior to those of the control group. Four patients in the UFT group received medication for 24 months and 14 patients were treated for more than 3 months. No severe adverse effects were observed. Seven patients suffered a relapse in the control group. Two patients suffered a relapse in the UFT group, but the relapse occurred after the discontinuation of UFT administration. We conclude that the administration of UFT as an adjuvant therapy prolonged the overall survival and disease-free survival rates of patients after the resection of NSCLC in a small study performed at a single institution. Interinstitutional differences, particularly operating procedures, should be carefully considered when performing large multicenter clinical studies.

Tumor-specific accumulation of 125I-labeled mouse-human chimeric anti-CEA antibody in a xenografted human cancer model demonstrated by whole-body autoradiography and immunostaining

Whole-body autoradiography (WBAR) was used to study the biodistribution of 125I-labeled mouse-human chimeric antibody (Ch F11-39) to carcinoembryonic antigen (CEA) in athymic nude mice bearing the CEA-producing MKN-45 human gastric carcinoma xenografts. Significantly high uptake of 125I-Ch F11-39 in the tumors obtained by tissue-counting technique was confirmed by WBAR of mice of 12, 24, 48, and 96 h postinjection of 125I-Ch F11-39. When compared with histochemical or immunohistochemical staining results of the tumor tissue sections, imaging profiles of 125I-Ch F11-39 obtained by WBARs were topographically correlated with histopathological findings of tissues and immunohistochemical localization of CEA in the tumor tissues, indicating that the accumulation of 125I-Ch F11-39 at the tumor site is based on its specificity for CEA. These results demonstrate that this chimeric antibody may serve as a potential useful diagnostic and/or therapeutic reagent for human CEA-producing cancers.

Molecular Identification of a Human Carcinoma‐associated Glycoprotein Antigen Recognized by Mouse Monoclonal Antibody FU‐MK‐1

Mouse monoclonal antibody FU‐MK‐1, raised against a human gastric adenocarcinoma, recognizes an antigen (termed MK‐1 antigen) present on the majority of carcinomas. The present study aimed to identify the MK‐1 molecule and to establish its relationship to other carcinoma antigens. Immunoprecipitation studies of human tumor cell lines revealed that FU‐MK‐1 recognizes a monomeric membrane glycoprotein with two forms, 40 kDa (major form) and 42 kDa (minor form), and with a molecular mass of 35 kDa following treatment with the N‐glycosylation inhibitor tunicamycin. The partial amino acid sequence of a main fragment of the MK‐1 molecule obtained by spontaneous cleavage under hypotonic conditions was examined, and the 17 contiguous NH2‐terminal amino acids were found to be identical with residues 81–97 of the 314‐residue GA733–2 protein [Szala et al.; Proc. Natl. Acad. Sci. USA, 87, 3542–3546 (1990)]. Hence, the GA733–2 cDNA was cloned and the specificity of FU‐MK‐1 was confirmed using four recombinant forms of the GA733–2 antigen expressed in COS‐1 cells. Immunoprecipitation with FU‐MK‐1 of the cell lysate transfected with the full‐length GA733–2 cDNA revealed two bands corresponding to those obtained from the tumor cell lines. FU‐MK‐1 also precipitated three other recombinant proteins consisting of amino acids 1–265, 1–201, and 1–139 of the GA733–2 protein, respectively. Furthermore, immunoblotting analysis indicated that FU‐MK‐1 binds to a small fragment (6 kDa) generated from a tumor cell line under hypotonic conditions, suggesting that the FU‐MK‐1 epitope exists on the distal 6‐kDa peptide of the extracellular domain of the GA733–2 molecule. We thus conclude that the MK‐1 antigen is the GA‐733–2 antigen, which is currently being used as a target in clinical trials with monoclonal antibodies.

Y-shaped tracheobronchial stent for carinal and distal tracheal stenosis.

A Y-shaped tracheo-bronchial tube was designed and used for two patients with carinal stenosis following a lower tracheal resection in one case and a malignant tracheal fistula in the other. The tube consisted of three parts including a Y-shaped, thin-walled, soft silicone stent; a spiral-wirereinforced main tube; and a curved tracheostomy tube. The stent was inserted easily and comfortably through the tracheostomy under fiberoptic bronchoscopic guidance with minimal local anesthesia. The positioning stability of the tube was excellent because of the carina-shaped structure of the tube end. Resistance to compression was satisfactory due to the embedded spiral wire. The insertion procedure through the tracheostomy was smooth, even in patients whose respiratory condition was severe or critical. Satisfactory phonational activity was also provided by breathing through the hole on the tube back up to the vocal cord. Bronchoscopic inspection was uncomplicated, and the patients themselves could easily clean the stent. Since palliation of the airway obstruction is the main purpose of such a stent for patients with either severe lower tracheal or carinal stenosis, and because of the difficulty of ordinary stent insertion in this part of the airway, this device appears to offer excellent stability and easy insertion of the stent. In addition, the ease of maintenance and suctioning through the tracheostomal end allows for an excellent quality of life in which the patients are able to return to their homes.

Effective purification of nonspecific cross-reacting antigens with phosphatidylinositol-specific phospholipase C

Two molecular species of nonspecific cross-reacting antigens, NCA-90 and NCA-50 with mol. wts. of 90,000 and 50,000, respectively, were effectively extracted with phosphatidylinositol-specific phospholipase C (PI-PLC) from human lung tissues, followed by extraction with perchloric acid, immunoaffinity chromatography with anti-NCA adsorbent, and gel filtration on a TSK G3000SW column. The yields of NCA were about 2 times more than those obtained by the usual method without PI-PLC. Addition of 0.05 unit of PI-PLC to 1 g of lung tissue and incubation at 37 degrees C for 1 h with continuous shaking seem to be practically sufficient for NCA extraction. The immunochemical properties of the NCAs thus obtained were found to be identical to those of NCAs obtained by the ordinary method.

Pulmonary papillary adenoma

We present a rare case of solitary pulmonary papillary adenoma. A man consulted our hospital because of abnormal chest radiography finding. Chest computed tomography demonstrated a well-defined, homogeneous nodular shadow 11 mm in size at the left lower lobe. The previous physician had considered it to be an old benign inflammatory granuloma and had kept it under observation. This mass was followed through chest radiographs at annual medical checkups for 4 years. In 2006, enlargement and lobulation were noted. We performed thoracoscopic partial resection of the left lower lobe. On postoperative pathology examination, the nodule was found to be a circumscribed nodule consisting of a papillary growth of cuboidal to low-columnar epithelial cells lining the surface of a fibrovascular stroma. The histological features were consistent with pulmonary papillary adenoma. Only 20 cases of pulmonary papillary adenoma have been reported in the literature.

A Rapid Colorimetric Assay for Carcinoembryonic Antigen (CEA)-Mediated Cell Adhesion and Analysis of CEA Domains Involved in the Adhesion

A colorimetric microadhesion assay that allows the quantitative determination of carcinoembryonic antigen (CEA)-mediated homophilic cell adhesion to CEA immobilized on 96-well polyvinyl chloride plates is described. Chinese hamster ovary (CHO) cells transfected with a full-length CEA cDNA were used as indicator cells. After dislodging nonadherent cells, specifically bound cells were quantified by a colorimetric analysis based on the ability of live cells to reduce the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product. The domains of CEA responsible for the homophilic cell adhesion were analyzed by inhibition assays using anti-CEA monoclonal antibodies whose reactive domains were already known. The involvement of domain N and possibly subdomain A3 of CEA in the homophilic cell adhesion has been suggested.