Use of SM-1 monoclonal antibody and human complement in selective killing of small cell carcinoma of the lung.

SM-1 is a murine monoclonal antibody strongly reactive with a cell membrane antigen of small cell carcinoma (SCC) of the lung but unreactive with the membrane of most other carcinomas and normal tissues including normal bone marrow. We have found that in the presence of human complement, SM-1 antibody is highly cytotoxic to SCC cells. Using three treatments with antibody and complement, more than 99% of SCC cells in culture were lysed, as determined by the chromium release and clonogenic assays. Similar efficiency of SCC cell lysis was observed when one SM-1 antibody treatment was followed by three treatments with human complement. In contrast, there was little antibody-dependent lysis of non-small cell lung cancer cells, other carcinomas, and leukemia cell lines. The amount of chromium released from normal bone marrow cells treated with SM-1 antibody and complement was minimal and was mainly due to the effect of complement alone. Clonogenic assays, including colony-forming unit-granulocytic/monocytic, erythroid burst-forming unit, and colony-forming unit-granulocytic/erythroid/monocytic/megakaryocytic, also showed no significant SM-1 antibody-dependent cytotoxicity on normal bone marrow precursors. Since SM-1 antibody is selectively cytotoxic to SCC cells in the presence of human complement, it is a potentially useful agent for the selective eradication of tumor cell contamination in marrows of patients with metastatic small cell lung cancer and possibly for in vivo serotherapy.

Membrane antigen in small cell carcinoma of the lung defined by monoclonal antibody SM1.

Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with a cell line derived from human small cell carcinoma (SCC) of the lung. The cloned hybridoma SM1 produced antibody that was reactive with the surface membrane of SCC cell lines and SCC tumors but not with the membrane of several non-SCC cell lines and tumors. SM1 ascites fluid was used to screen for reactivity of the antibody with other human cancer cell lines, tumor tissues, and normal tissues. SM1 antibody was found to be unreactive with neuroblastoma, adrenal carcinoma, melanoma, and bronchial carcinoid. Reactivity was detected with some breast carcinoma cell lines but not with breast cancer tissue specimens. In the same individual, the antibody was reactive with SCC lung tumor and SCC metastatic to the liver but not with normal tissues, including bronchus, lung parenchyma, liver, kidney, and brain. Human erythrocytes and marrow cells were also unreactive. Since SM1 detects an antigen that is present in greatest amounts on the surface membrane of SCC of the lung, this antibody may be useful in tracing the lineage patterns of human lung cancers.

Selective cytotoxicity of the SM1 monoclonal antibody towards small cell carcinoma of the lung.

A murine monoclonal antibody, SM1, is strongly reactive with the surface membrane of small cell carcinoma of the lung. SM1 antibody is unreactive with most other cancers and various normal tissues including bone marrow cells. We now find that SM1 antibody is selectively cytotoxic to small cell carcinoma (SCC) in vitro. The antibody is present in high titers in supernatant fluids or ascites obtained by i.p. injection of SM1 hybridoma cells into pristaned BALB/c mice. The cytotoxic effect of the antibody is reduced to one-half maximal activity only at dilutions greater than 1:40,000. The efficiency of tumor cell lysis is greatly enhanced by repeated treatments with antibody and complement. Using three treatments with antibody and complement, 99.9% of SCC cells are lysed, as determined by the chromium release. Similar efficiency of SCC cell kill was observed by clonogenic assays. SM1 antibody produces no significant antibody-dependent lysis of cell lines derived from non-SCC lung carcinomas and leukemia cells. The results from chromium release assay and clonogenic assays also indicate that the effect of SM1 antibody and complement on bone marrow cells is minimal and could be accounted for by the effect of complement alone.

Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung.

The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin-embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen.

Detection of bone marrow metastasis in small-cell lung cancer by monoclonal antibody.

A murine monoclonal antibody against a surface antigen of small-cell carcinoma of the lung (SM1 antibody) was investigated for its use in detecting bone marrow metastasis. Bone marrow cells of healthy volunteers and of patients with small-cell carcinoma of the lung (SCCL) were examined for reactivity with SM1 antibody and indirect immunofluorescence and the results compared to conventional histochemical staining (Wright-Giemsa stain of bone marrow aspirates and hematoxylin-eosin stains of bone marrow biopsies). No SM1 reactivity was found in marrow cells of eight healthy volunteers. Thirty-six samples from 33 patients with SCCL were examined; tumor involvement was found in 69% by SM1 antibody and in 16% by histochemical stains. All bone marrow samples from patients with SCCL that were unreactive with SM1 antibody also showed no evidence of tumor involvement by histochemical stains. Samples of 29 patients were investigated at initial staging; SM1 reactive cells were found in 50% of 16 patients with limited disease and in 77% of 13 patients with extensive disease. Overall, the proportion of patients recognized to have disseminated disease at diagnosis was increased from 45% to 72% by monoclonal antibody staining. Indirect immunofluorescence with SM1 antibody allows detection of bone marrow metastasis of SCCL that cannot be seen by conventional morphology and can identify disseminated disease in patients otherwise staged limited disease.

Localization of radiolabeled SM1 monoclonal antibody to small-cell lung cancer tumors in mice.

Small-cell carcinoma (SCCL) is an aggressive type of lung cancer. Though it is usually responsive to therapy, be it chemotherapy or radiation, the majority of patients eventually relapse and overall prognosis is dismal. New forms of therapy are, therefore, needed. SM1 monoclonal antibody (MAb) was developed in our laboratory and demonstrated to be highly reactive against SCCL. I125 radiolabeled SM1 antibody was administered intravenously to nude mice bearing SCCL tumor xenografts. The mice were sacrificed, different tissues sampled and tested for uptake of radioactivity five days following antibody injection. There was over a 30 fold increase in localization of labeled antibody to the tumor as compared to muscle tissue. All organs tested showed an insignificant amount of MAb (p = 0.01) including the spleen, which had the highest normal tissue uptake in these experiments. These results demonstrate that SM1 MAb can be successfully targeted to SCCL xenografts. Its potential applications for imaging and therapy of SCCL in man are currently under investigation.

Murine monoclonal antibody LAM2 defines cell membrane determinant with preferential expression on human lung small-cell carcinoma and squamous-cell carcinomas.

Murine monoclonal antibodies (MAbs) were generated against a human undifferentiated lung carcinoma cell line. The hybridoma designated LAM2 produced an IgM kappa MAb with reactivity to the cell membrane. Indirect immunofluorescence staining and radioimmunoassay showed LAM2 antibody to react preferentially with lung small-cell carcinoma (SCC) cell lines and squamous-cell carcinoma (SQC) cell lines. LAM2 antibody also stained primary cultures of normal bronchial epithelial cells, but was unreactive with human erythrocytes and nucleated marrow cells. Indirect immunofluorescent staining with LAM2 antibody was performed on frozen sections of human tumor tissues and normal tissues. LAM2 antibody stained all 8 SCC carcinomas, 4 of 5 SQC of the lung and head and neck region, and 2 or 4 lung large-cell carcinomas. No staining was seen on lung adenocarcinomas, breast carcinomas, ovarian carcinomas, renal cell carcinomas, colon carcinomas, or mesotheliomas. Staining was present on sections of normal bronchus, but not on normal lung parenchyma, liver, kidney, adrenal or skin. While LAM2 antibody was highly reactive with all SCC examined, its antigenic determinant was not expressed in other cell lines and tumors of presumed neuroectodermal origin, including neuroblastoma, melanoma, and bronchial carcinoid. Radioimmunoprecipitation showed the antigen defined by LAM2 antibody to have two major bands of approximate molecular weights of 45,000 and 125,000. The selective reactivity of LAM2 antibody with SCC and SQC, but not with most other tumor tissues and normal tissues, makes it a good candidate for use in clinical diagnosis and possibly serotherapy.

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.

SQM1 antibody defines a surface membrane antigen in squamous carcinoma of the head and neck.

We produced murine monoclonal antibodies (MAbs) directed against the surface membrane of squamous-cell carcinoma of the head and neck (SCCHN). One antibody, SQM1, was determined by immunofluorescence and radioimmunoassay to be reactive with 13/13 SCCHN cell lines derived from different sites of the head and neck area. No binding reaction was observed with normal fibroblasts, red blood cells, nucleated bone-marrow cells or epithelial cells from normal oral mucosa. SQM1 reactivity was observed with primary cultures of normal epidermal and bronchial epithelial cells. Significant reactivity was found with 2/4 cell lines derived from small-cell carcinoma of the lung but little or no reactivity was found with other lung cancer cell lines. Various cell lines derived from other cancers including breast, colon, and ovarian carcinomas, melanoma, neuroblastoma and leukemia were generally unreactive. Seventeen out of 18 fresh frozen specimens of SCCHN were strongly reactive with SQM1 antibody. However, autopsy specimens from the heart, liver, kidney, spleen, colon, subcutaneous fat and skin connective tissue were unreactive. SQM1 antibody may be useful in biological and clinical studies of the head and neck region.

Reduced membrane protein associated with resistance of human squamous carcinoma cells to methotrexate and cis-platinum.

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SqCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SqCHN cells which developed resistance to the alkylating drug cis-latinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.