A monoclonal antibody KIS-1 recognizing a new membrane antigen on human squamous-cell carcinoma

A KIS-1 monoclonal antibody (MAb) (IgG₁, K) recognizing a membrane antigen on human squamous-cell carcinomas (SCC) was developed to understand their antigenicity using an esophageal SCC as an immunogen. The KIS-1 MAb recognized a membrane antigen on a majority of esophageal, lung, and oral-cavity SCC by immunofluorescent and by immunohistochemical analyses. In contrast, it showed little reactivity to adenocarcinomas from different organs, and none to keratinocyte cell lines. This MAb showed reactivity to the cells in the basal layer of normal esophageal epithelium adjacent to the esophageal SCC, but none of the other normal tissues, including esophageal epithelium far from the SCC and that from patients with non-malignant disease. The KIS-1 MAb immunoprecipitated a 46-kDa membrane protein of the esophageal SCC in non-reducing and in reducing conditions. It recognized the 46- and the 40-kDa proteins of the esophageal SCC by immunoblot analysis. These results suggest that the KIS-1 MAb recognizes a new membrane antigen preferentially expressed on SCC, and that this antigenicity is shared only by the cells in the basal layer of the esophageal epithelium adjacent to SCC. The KIS-1 MAb may be a new tool for understanding the antigenicity of SCC. © 1996 Wiley-Liss, Inc.     

Tumor-associated membrane sialoglycoprotein on human small cell lung carcinoma identified by the IgG2a monoclonal antibody SWA20

A mouse IgG2a monoclonal antibody, SWA20, defining a tumor-associated cell surface antigen on small cell carcinoma of the lung (SCC) was generated. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and solid phase radioimmunoassay and the reactivity with tissues by immunoperoxidase staining. The antibody reacts with a proportion of small cell carcinoma cell lines (4 of 8) and tissues (7 of 12), but not with other pulmonary or extrapulmonary cell lines (0 of 30) or tumor tissues (0 of 78). The antibody was unreactive with primary cultures of normal bronchial epithelial cells, RBC, and WBC. Immunoperoxidase staining of normal tissues showed rare antigen-positive cells in suprabasal layers of bronchial epithelium and less than 10% of positive cells in colon epithelium. Immunoblots of SCC extracts demonstrated antibody reactivity with a doublet band at Mr 40,000, a broader band at Mr 100,000, and a band at Mr 180,000. The antigen was not present in crude lipid extracts of SCC cells. Solid phase radioimmunoassays and immunoblots showed binding competition with the lectin Triticum vulgaris, sensitivity of the antigen to neuraminidase, and a partial sensitivity to treatment with periodate. The antigen was coexpressed on SCC cell lines with the antigen sGP90-135 defined first by antibody LAM8 (R. Waibel, C. J. O'Hara, and R. A. Stahel. Cancer Res., 47:3766-3770, 1987) but differed from it by lack of reactivity with Lea-positive saliva and partial resistance to periodate treatment. There was no binding competition between radiolabeled antibodies SWA20 and LAM8 to SCC target cells. The IgG2a antibody SWA20 identifies a previously undescribed tumor-associated surface membrane antigen, sGP100, expressed selectively on a proportion of SCC.     

Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody

Acetone-fixed frozen tissue sections from 56 cases of human lung carcinoma were tested for reactivity by an indirect immunoperoxidase technique with a monoclonal antibody (MAb 528) specific for the external domain of the epidermal growth factor receptor (EGFR). MAb 528 reacted with all epidermoid (22/22) and large-cell (4/4) lung carcinomas evaluated. The antibody was also positive with a subset of lung adenocarcinomas (13/21) and did not react with small-cell lung cancers (SCLCs) (0/9). MAb 528 also stained normal bronchial epithelium identified within the tumor sections of 5 cases. Thus EGFR was expressed by all epidermoid and large-cell lung carcinomas examined, a subset of lung adenocarcinomas, and normal bronchial epithelium. EGFR expression was not identified in any of the SCLCs tested. These data imply that immunohistochemical detection of EGFR expression may find future application in distinguishing epidermoid, large-cell, and some adenocarcinomas of the lung from SCLCs.     

Selective cytotoxicity of murine monoclonal antibody LAM2 against human small-cell carcinoma in the presence of human complement: possible use for in vitro elimination of tumor cells from bone marrow

LAM2 is a murine IgM monoclonal antibody (MAb) which binds strongly to the cell membrane of human lung small-cell carcinoma (SCC) and squamous-cell carcinoma but not to normal bone-marrow cells. The cytotoxicity of this antibody in the presence of human complement was investigated in vitro by chromium release and clonogenic assays. The optimal treatment conditions included incubation with antibody for 30 min at 37 degrees C followed by 3 additions of human complement 30 min apart. Cell lysis ranged from 94 to 98% in 4 SCC cell lines at antibody dilutions of 1:100: a lower level of lysis (60%) occurred in a lung squamous-cell carcinoma cell line. The cytotoxic effect was strictly complement-dependent. No cytotoxic effect was seen with other human cell lines including lung adenocarcinoma, lung large-cell carcinoma, myeloid leukemia, and lymphoblastic leukemia. No lysis was seen with nucleated marrow cells from healthy volunteers. Normal marrow cells in excess did not inhibit SCC cell lysis. Incubation with antibody and complement resulted in a 100-fold reduction of colony formation of SCC cells, but did not reduce the number of colonies of marrow-cell precursors, including CFU-GEMM, BFU-E, and CFU-C. The selective cytotoxicity of LAM2 antibody to SCC, but not to normal bone-marrow cells, suggests that this antibody may be useful for the in vitro elimination of SCC cells from the bone marrow.     

Murine monoclonal antibodies raised against human non-small cell carcinoma of the lung: specificity and tumor targeting

The tumor targeting properties of murine monoclonal antibodies (MAbs) generated in our laboratory against non-small cell carcinoma of the lung have been investigated in nude mouse xenograft models. The MAbs selected for evaluation, RS5-4H6, RS7-3G11, and R511-51, have pancarcinoma reactivity, as shown by immunoperoxidase staining of the majority of tumors from the lung as well as breast, colon, kidney, and ovary. The localization of the three MAbs which bind to distinct antigens, and exhibit different levels of cross-reactivity with normal human epithelial tissues, are compared. The MAbs are of the IgG1 isotype. Since these MAbs were reactive with Calu-3, a human adenocarcinoma of the lung cell line grown as xenografts in nude mice, this system was selected as our initial tumor target. The MAbs were found to localize preferentially to the heterotransplanted tumors, with from 6.6 to 8.6% of the injected dose per gram accreting in the tumor at 7 days. Tumor/nontumor ratios of up to 9.7 were seen with one MAb at day 14. The targeting of MAb RS11-51 and F(ab')2 fragments of RS11-51 in GW-39, a human colon cancer grown in nude mice, was also studied. Accretion of intact RS11-51 and F(ab')2 fragments into GW-39 was greatly increased compared to Calu-3. In view of the high frequency of antigen expression on a wide variety of tumors, and the ability to target in vivo, these new MAbs may have potential use in the imaging and therapy of cancer.     

Association of p53 expression with proliferative activity and poor-prognosis in patients with squamous-cell lung carcinomas

Tumor tissue of 65 patients with squamous cell lung carcinoma was analyzed for p53 expression using immunohistochemistry (DO-1) and for proliferative activity using flow cytometry. Of the 65 cases, 17 cases (26%) showed positive staining for p53, whereas 48 cases (74%) showed no expression. The median survival time for patients with p53-negative tumors was 100 weeks and for patients with p53-positive tumors 30 weeks (rank-sum test, p=0.03; log-rank test, p=0.14). The median survival time for patients with high proliferative activity (proportion of SG(2)M-phase cells >22%) was one year and for patients with low proliferative activity (proportion of SG(2)M-phase cells less than or equal to 22%) over 6 years (rank-sum test, p=0.04; log-rank test, p=0.01). There exists a trend that p53-positive squamous cell lung carcinoma had a higher proportion of SG(2)M-phase cells than p53-negative tumors. Multivariate analysis found independent prognostic significance for proliferative activity and stage but not for p53.     

Glycoproteins distinguishing non-small cell from small cell human lung carcinoma recognized by monoclonal antibody 43-9F

Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6] and mucin-like properties. A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate. The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells. A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell. This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen. In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands. In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells. Other organ systems lack any reactive cells. The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope. The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.     

Molecular basis for lack of expression of HLA class I antigens in human small-cell lung carcinoma cell lines

HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and thus play an important role in immune surveillance of cells infected with virus or altered by malignant transformation. Immunochemical studies have demonstrated a marked deficiency or lack of expression of class I molecules on the surface of many different types of tumor cells. It is likely that this allows these cells to escape immune surveillance. In the present study, we examined the molecular basis for lack of expression of class I antigens in small-cell lung carcinoma cell lines. Our results demonstrate that these cell lines also lacked products of MHC-encoded proteasome subunit LMP2 and the putative peptide transporter TAP1. In contrast, LMP7 and TAP2 genes were expressed in these cell lines. Pulse-chase experiments showed that class I molecules were unstable and thus not transported to the cell surface from endoplasmic reticulum. Our results suggest that antigenic peptides were not available for binding to class I &agr: chains due to genes showed that the tumor cells lacked trans-regulatory nuclear protein(s), which binds to the interferon-γ (IFN-γ) response element (ISRE) in the TAP I, LMP2 bidirectional intergenic promoter. Treatment of tumor cells with IFN-γ induced ISRE-binding nuclear protein(s) and resulted in expression of TAP 1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. Our data provide credence for a role of TAP and LMP genes in immune response. © 1996 Wiley-Liss, Inc.     

A monoclonal antibody reacting with various human carcinomas and fetal colon and esophagus

A monoclonal antibody 9A3 was raised to human gastric carcinoma cell line TMK-1, a poorly differentiated adenocarcinoma of the stomach. 9A3 antibody (IgG1, kappa) strongly reacted with various carcinomas immunohistochemically, but did not react with any normal tissues of the whole body tested except for neutrophiles and macrophages. 9A3 antibody also reacted with the luminal surface of the fetal colon and esophagus at six months of gestation. The antigen recognized by 9A3 antibody might be an oncodevelopmental antigen.     

Antibody dependent cell mediated cytotoxicity on human cervical carcinoma cell line, ME-180, with human monoclonal antibody.

A human monoclonal antibody (MCA), CLN-IgG, showed cytotoxic effect in vitro against the cervical carcinoma cell line, ME-180, by antibody dependent cell-mediated cytotoxicity (ADCC). To determine which fractions of cells in peripheral blood lymphocyte (PBL) mediate ADCC, PBL were separated with nylon wool column and sheep red blood cells (SRBC). Both adherent cells (monocyte) and non-T, non-B cells showed cytotoxicity by ADCC. Human non-T, non-B cells showed higher cytotoxic activity against ME-180 cells than monocytes. Furthermore murine effector cells were less effective in ADCC than human effector cells with human MCA.     

A monoclonal antibody (B72.3) defines patterns of distribution of a novel tumor-associated antigen in human mammary carcinoma cell populations

We report here both the range and patterns of reactivity of an IgG1 monoclonal antibody, B72.3, prepared against human, metastatic mammary carcinoma cells. When the avidin-biotin complex (ABC) immunoperoxidase technique was used on tissue sections, monoclonal B72.3 reacted with 19 of 41 (46%) primary mammary carcinomas and 13 of 21 (62%) metastatic lesions, either in axillary lymph nodes or at distal sites. Variable concentrations of antigen, recognized by B72.3, were observed among mammary tumors, as well as among different cell populations of a given tumor mass. Several patterns of antigen distribution were observed: membrane, diffuse cytoplasmic, focal and marginal. No reactivity was observed to normal mammary epithelium, stroma, or lymphocytes of the breast, nor to any cell types in a variety of other normal human tissues, melanomas, and sarcomas. Reactivity with all of four colon carcinomas was also observed. Assay of serial sections of mammary carcinomas with B72.3 and a monoclonal antibody directed against carcinoembryonic antigen demonstrated that these antigens were both distinct and non-coordinately expressed.     

Comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma

Expression profiling by gene microarray techniques have been developed to predict malignant tissue but there are no experiences with the application of antibody arrays to identify malignancy-related proteins. Because altered protein patterns might also better interpret biological processes, we applied tumour samples from 12 patients with squamous cell lung carcinoma and individual lung tissue controls to antibody arrays spotted with 378 distinct monoclonal antibodies. Array analysis defined 20 proteins with higher and nine with lower abundance in lung tumours. Comparison with gene microarray data revealed that 31% of the differentially regulated proteins correlate with altered mRNA expression in squamous cell lung carcinomas, including PEX1, MKK7 and HDAC3 for up-regulated proteins. The histone deacetylase (HDAC) 3 was investigated in detail by immunoblot analysis showing that HDAC3 is indeed elevated in 92% of tumours (n=22/24; P<0.001). Thus, antibody microarrays can be useful for detection of some target proteins related to lung cancer.     

Immunoscintigraphy of human lung squamous cell carcinoma using an iodine-131 labelled monoclonal antibody (Po66)

Monoclonal antibody (McAb) Po66 has been obtained by immunisation of mice against a human lung squamous cell carcinoma. The in vitro reactivity of the antibody with cancer cells and its ability to localise in human lung cancer xenografts growing in nude mice have been reported earlier. Presented here is the first clinical evaluation of the antibody for scintigraphic detection of tumours. Thirty-three patients with histologically confirmed primary non-small cell lung carcinoma were investigated. Twenty-seven of them were explored at the preoperative stage and six at 6 months after surgery. Biodistribution results were obtained from seven operated patients by combining injections of 131I-radiolabelled Po66 and of 125I-labelled unrelated immunoglobulin. The localisation index was three times higher for this specific antibody. Immunoscintigraphy detected 78% of primary tumours and 100% of recurrences. In this short series of patients, immunoscintigraphy proved helpful in the assessment of tumour spread in four patients by visualising localisations in the mediastinum or the contralateral lung which the CT scan had failed to demonstrate. Immunoscintigraphy was also more efficient than plain chest X-ray for the detection of local tumour recurrences.     

Identification of human acinar cell carcinoma by monoclonal antibody and in vitro differentiation

Methylnitrosourea (MNU)-induced carcinomas in organ-cultured human pancreas when injected into nude mice produced subcutaneous carcinomas none of which were recognizable as being of acinar cell origin. Both monoclonal antibody to acinar cell surface marker produced by hybridoma, and in vitro tumor cell differentiation were used to detect tumors of acinar cell origin. Only 1 out of 14 tumors, a highly undifferentiated carcinoma, proved to be of acinar origin. This tumor was composed of cells devoid of zymogen granules but with abundant acinar cell surface marker. The acinar origin of this tumor was also confirmed by its differentiative features after 7 weeks of culture.     

Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody.

The chimeric antiganglioside GM2 monoclonal antibody (MAb) KM966, which showed high effector functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), potently suppressed growth and metastases of GM2-positive human cancer cells inoculated into mice. To further improve the therapeutic efficacy of the anti-GM2 MAb in humans, we constructed a humanized anti-GM2 MAb, KM8969. The humanized KM8969 was more efficient in supporting ADCC against GM2-positive human cancer cell lines than the chimeric KM966, whereas complement-dependent cytotoxicity was slightly reduced in the humanized KM8969. In addition, the humanized KM8969 was shown to exert potent ADCC mediated by both lymphocytes and monocytes. To investigate the effect of the humanized KM8969 on the biological function of GM2 in the condition physiologically mimicking formation and growth of cancer masses, the heterospheroids composed of normal human dermal fibroblasts and GM2-positive human lung cancer cells were developed. Interestingly, the humanized KM8969 gave rise to growth inhibition of heterospheroids without dependence of the effector functions. Morphological and immunocytochemical analysis suggested that the inhibitory effect was due to the apoptosis of GM2-positive cancer cells in the heterospheroids. The result indicates that GM2 captured by the antibody on the cell surface loses its physiological function that plays a critical role in maintaining the three-dimensional growth of cancer cells in contact with its own cells or other type of cells in a microenvironment. The humanized KM8969, which can destroy the cancer cells via blocking functional GM2 on the cell surface as well as the effector functions, would have extraordinary potential in human cancer therapy.