Monoclonal antibody SEN7 recognizes a new epitope on the neural cell adhesion molecule present on small cell lung cancer but not on lymphocytes.

In our continuing attempt to select monoclonal antibodies for immunotargeting of small cell lung carcinoma (SCLC) we have developed the IgG1 murine antibody SEN7 which by immunofluorescence stained all SCLC cell lines tested. On frozen tumor section six of seven SCLCs were positively stained. The reactivity of this antibody in a series of lung tumors and on normal tissues has some similarities with cluster 1 antibodies and cluster w4 antibodies, as defined by the First and Second International Workshop on Lung Cancer Antigens [P.C.L. Beverley, Y. Olabrian, J.A. Ledermann, L.G. Bobrow, and R.L. Souhami, Br. J. Cancer, 63 (Suppl): 10-19, 1991], particularly with regard to staining of neuroendocrine tissues. The similarities in staining of neuroendocrine tissues between antibody SEN7 and cluster 1 and cluster w4 antibodies prompted us to examine the binding of SEN7 with transfectants expressing the respective antigens. On the murine lymphoma cells B-9, stably transfected with a complementary DNA clone coding for an M(r) 140,000 isoform of human SCLC neural cell adhesion molecule (NCAM), antibody SEN7 reacted positively whereas the cluster w4 antibody was negative. The reaction of antibody SEN7 with the NCAM transfected murine lymphoma cells was unexpected in view of its lack of binding to peripheral blood mononuclear cells which regularly stain positive with NCAM antibodies. Western blotting of a renatured SCLC extract revealed a strong band around M(r) 180,000 in contrast to other cluster 1 antibodies which recognized a broad polydisperse band with a molecular weight of 140,000 to 210,000. Antibody binding was sensitive to tunicamycin treatment, suggesting the epitope to reside on an N-linked carbohydrate structure. No significant competition for SEN7 binding on SCLC cells was seen with other NCAM antibodies against the three distinct epitopes described on SCLC. This finding together with the lack of staining of peripheral blood mononuclear cells and the selected reactivity with the M(r) 180,000 band of NCAM indicate the antibody SEN7 recognizes an epitope on NCAM which has not been described previously. Biodistribution studies with radiolabeled SEN7 in nude mice bearing s.c. SCLC xenografts demonstrated the selective localization of more than 30% of the total injected dose per g tissue at day 4 following i.v. injection. The homogeneous binding to SCLC, the lack of binding to peripheral blood mononuclear cells, and the favorable tumor localization in a xenograft model indicates that SEN7 is a good antibody for immunotargeting of SCLC.

BSPRY, a novel protein of the Ro-Ret family.

Utilizing the yeast two-hybrid system we have identified a novel protein of the Ro-Ret family that was termed BSPRY. This protein is composed of a B-box, an alpha-helical coiled coil and a SPRY domain. BSPRY from human beings shares 80% sequence identity with the homologous protein from mice. The gene for BSPRY resides on human chromosome 9 and is specifically expressed in testis. It comprises six exons and five introns and possesses a GC rich promoter forming a typical CpG island. The function of BSPRY is not known, but several related proteins of the RBCC family have been implicated in cell transformation.

Reactivity of monoclonal antibodies directed against lung cancer antigens with human lung, breast and colon cancer cell lines.

A panel of monoclonal antibodies (n = 72 including controls) directed against lung cancer antigens was screened immunohistochemically against a panel of seven human lung cancer cell lines (including small cell carcinoma, squamous cell carcinoma, adenocarcinoma and mesothelioma), six human breast cancer cell lines and one human colon cancer cell line. The majority of the antibodies (n = 42) reacted also with antigens present on breast and colon cancer cell lines. This cross reactivity especially between lung and breast cancer cell lines is not altogether unexpected since antigens common to breast and lung tissue including their neoplasms such as MUC1 antigen have been described. Our results indicate that epitopes shared by lung and breast cancers are probably more common than previously thought. The relevance for prognosis and therapy of these shared antigens, especially as disease markers in breast cancer, has to be investigated.

Assisted suicide as conducted by a "Right-to-Die"-society in Switzerland: a descriptive analysis of 43 consecutive cases.

BACKGROUND AND METHODS: The Swiss "Right-to-Die"-society EXIT enables assisted suicide by providing terminally ill members with a lethal dosage of barbiturates on request. This practice is tolerated by Swiss legislation. EXIT insists on its assumption that people with serious illness and suffering have the competency to take such a decision. The case of two patients who committed suicide a short time after their release from a psychiatric clinic raised some doubts about the practice of EXIT. The files of all 43 cases of suicide assisted by EXIT between 1992 and 1997 in the region of Basle kept in the Institute of Forensic Medicine were examined for accuracy of the medical data. This sample was compared for age, gender-ratio and prior psychiatric treatment with 425 ordinary suicides in the same region. An attempt was made to assess whether only terminally ill and people with intolerable suffering had been assisted with suicide and what efforts EXIT had made to rule out psychiatric illnesses or poor social conditions as the reason for the wish to die. RESULTS: A medical report of the treating doctor(s) was in the files in only five cases. The "EXIT" cases where older than the "ordinary"-sample. Among those over 65 years old there were almost twice as many women as men. 16 of the 24 women older than 65 years were widowed. There were 20 cases of cancer; but in eleven cases medical files revealed no apparent medical condition to explain a death-wish. Five of the patients declared a social loss or fear of such loss as the reason for their wish to die. Six persons had formerly been in psychiatric care, though this was not mentioned in the files. CONCLUSIONS: Due to the scarcity of information in the files as regards previous palliative care, the high proportion of old women and the high percentage of people not suffering from a terminal illness compared to the literature we conclude that psychiatric or social factors are not an obstacle for EXIT to assist with suicide.

Down-regulated proteins of mesenchymal tumor cells.

To identify proteins that are lost during the establishment of the transformed phenotype of a tumor cell, we have prepared a subtracted cDNA library with mRNA from normal human fibroblasts and from their matched SV40 transformed counterparts. More than 40 clones were obtained that showed a dramatic reduction in their relative expression after oncogenic transformation. The proteins encoded by these clones could be grouped into four distinct classes: extracellular matrix proteins (fibronectin, beta ig-h3, collagen VI), enzymes (collagenase, urokinase), cytoskeletal proteins (vinculin, SM22) and regulatory proteins (beta-glycan, integrin-associated protein, myosin kinase, IGFBP-5). Six novel gene products were discovered during these experiments, including a novel serine protease, a zyxin-like protein, an ankyrin-like protein and a GTP-binding protein. Only four of all the transformation-sensitive cDNAs were consistently down-regulated when a variety of cell lines derived from spontaneous mesenchymal tumors was investigated: beta ig-h3, collagen VI, the novel ankyrin-like protein, and IGFBP-5. It is likely that these gene products play an important role in the maintenance of the normal phenotype.

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

Blood units for patients with irregular blood group antibodies: frequency and supply.

Blood banks face significant problems supplying blood units compatible with erythrocyte antigens other than ABO and rhesus D. In this study the number of such units was evaluated between 1973 and 1982, and it was found that 1.5% of the total number of blood units were compatible with other antigens than ABO and rhesus D. Based on data of the number of units requested for each such antigen and the frequency of the antigen in the European population, a logistical system providing sufficient numbers of 'antibody-compatible' blood units is proposed.

Characterization of human matrilin-3 (MATN3).

We have isolated a cDNA clone for human matrilin-3 from a cartilage-specific cDNA library. The polypeptide predicted from the nucleotide sequence of this clone shared 83% identity with matrilin-3 from mouse and 61% with that from chicken. It was composed of 486 amino acid residues that were arranged in seven domains: a signal peptide, a von Willebrand factor A domain, four EGF repeats, and an alpha-helical region. The gene for human matrilin-3 (MATN3) was assigned to chromosome region 2p24-p23. The corresponding mRNA of 2.8 kb was expressed in every type of cartilage investigated thus far. It was also produced in vitro by primary chondrocytes isolated from articular cartilage. However, dedifferentiated chondrocytes of the third passage did not express it at all. Matrilin-3 might therefore serve as a marker for the differentiation state of chondrocytes.

[Assisted suicide and psychiatric disorders]. / Beihilfe zum Suizid bei psychisch Kranken.

In the region of Basel in Switzerland 43 assisted suicides were registered between 1992 and 1997, eight percent of all suicides in the region. Assisted suicide was administered by the help-to-die society Exit. There was a psychiatric history in six of the suicides. Four of them suffered of serious physical illness as well. The analyses of these six suicides focuses on the conditions of assisted suicide in people with mental illness and the ethical problems arising.

[Acanthocytosis in chronic septic granulomatosis: the McLeod syndrome]. / Akanthozytose bei septischer Granulomatose: McLeod-Syndrom.

Acanthocytosis was observed in a boy suffering from Chronic Granulomatous Disease (CGD). Further investigations revealed weak Kell-antigen-expression on the patient's erythrocytes. This so-called "McLeod-Syndrome" is due to the absence of Kx, the Kell antigen precursor substance. So far, 8 cases of an association between McLeod-Syndrome and CGD have been reported. Both genetic defects are closely linked on the X-chromosome and may therefore be inherited together. In female carriers, the variable inactivation of one X-chromosome according to the Lyon-hypothesis is the reason for the appearance of both normal and McLeod-erythrocytes at the same time. This was observed in the mother and sister of our patient too. Blood transfusions should be avoided, because McLeod-patients are at risk to form antibodies against the precursor substance Kx common to the erythrocytes of virtually all people. These patients should therefore be encouraged to donate blood which can be stored frozen and used either as autotransfusion (for the patient himself) or for other McLeod-patients.

A novel GTP-binding protein which is selectively repressed in SV40 transformed fibroblasts.

We have used a subtractive hybridization procedure to isolate cDNA clones for proteins that are produced by human fibroblasts, but not by their SV40-transformed counterparts. With this technique we found, in addition to fibronectin and collagen VI, a novel GTP-binding protein. Sequencing of overlapping cDNA clones demonstrated that this protein is composed of 364 amino acids with a molecular mass of 41 kDa and a calculated isoelectric point of 9.4. It contains the five sequence motifs G1-G5 that are conserved in all GTP-binding proteins. Apart from these characteristic motifs the amino acid sequence differs substantially from those of the well characterized G-proteins, but it is similar to those of some recently identified proteins from Caenorhabditis elegans, from Schizosaccharomyces pombe, and from an archaebacterium, suggesting the existence of a new subfamily within the superfamily of the GTP-binding proteins. The striking conservation of the primary structure between distantly related species indicates a fundamental function of the new protein. Since it is produced in normal, but not in virally transformed fibroblasts, it may play a role in the expression of the transformed phenotype or in growth control.

Expression of alternatively spliced forms of the CD44 extracellular-matrix receptor on human lung carcinomas.

Expression of isoforms of the CD44 hyaluronan receptor/lymph-node endothelial receptor by human tumour cells is thought to play a role in tumour growth and metastasis. These isoforms which vary in the length of the extracellular domain are generated by differential RNA splicing that involves the 10 alternative exons (v1 to v10) encoding the membrane proximal region of the molecule. Several tumours have been shown to over-express CD44 containing the v6 exon, and this, together with other evidence, has led to the suggestion that v6 may play a causative role in tumour metastasis. In this report we have compared the expression of CD44 isoforms between different lung tumour lines, including SCLC, squamous-cell carcinoma, adenocarcinoma and mesothelioma, using both RT-PCR and fluorescent antibody staining with a panel of CD44 exon-specific monoclonal antibodies (MAbs). Our results show large differences in vCD44 expression between individual tumour lines. Little or no vCD44 containing the metastasis-associated v6 exon was detected in most tumours, including the highly metastatic SCLC lines. Indeed, the SCLC lines and some squamous-cell carcinomas contained only very low levels of either vCD44 or CD44H, indicating that CD44 expression may not always correlate with tumour development or dissemination. One of the squamous-cell carcinomas studied (HOTZ) was found to express a complex mixture of CD44 splice variants similar to the immortalized normal bronchial epithelial line BEAS-2B. Cloning and sequencing of vCD44 from the HOTZ cell line yielded several splice variants that have also been identified on leukaemic cells, normal keratinocytes and activated peripheral-blood lymphocytes.

Immunological evidence for co-expression of cluster-5 and cluster-5A small cell lung cancer antigen on a single molecule.

We have investigated immunologically the molecular association of the cell-surface sialoglycoprotein antigens cluster-5 (CL-5) and cluster-5A (CL-5A), known to be co-expressed in human small-cell lung cancer (SCLC). CL-5 antigen is exclusively defined by IgM antibodies as represented by MAb LAM8, whereas CL-5A antigen is exclusively defined by IgG antibodies as represented by MAbs SWA20 and SEN31. Because of the unavailability of purified antigens, the question of the molecular relationship between these antigens was addressed by immunological studies. We generated an anti-anti-idiotypic MAb of the IgG isotype using a CL-5-antigen-mimicking anti-idiotype defined by rat MAb Ly8-229 as an immunogen to circumvent the avidity problems observed with the IgM MAb LAM8 in binding-competition experiments. In addition, we developed a heterologous double antibody sandwich assay able to identify circulating CL-5/5A antigens in pre-treatment sera of patients with SCLC. The results of both types of immunological studies demonstrated the expression of CL-5 and CL-5A antigens on a single molecule, both in cellular assays and in assays detecting antigens shed into the serum of patients with SCLC.

Statistical analysis of data from the Third International IALSC Workshop on Lung Tumor and Differentiation Antigens.

The main aim of the statistical analysis of data collected in the Third International IALSC Workshop on Lung Tumor and Differentiation Antigens, was to identify groups of monoclonal antibodies (MAbs) having similar profiles of reactivity against a variety of cell types in flow cytometry, histology, immunofluorescence and immunocytochemistry experiments. This was achieved through cluster analysis. We describe the methods used in the cluster analysis, and in the data processing leading to it.

Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines.

Expression of Bcl-2 is life-sustaining for small-cell lung cancer cells and associated with drug resistance. In the present study, the interactions between the bcl-2 antisense oligodeoxynucleotide 2009 and the chemotherapeutic agents etoposide, doxorubicin and cisplatin were investigated on small-cell lung cancer cell lines to search for synergistic combinations. The cell lines NCI-H69, SW2 and NCI-H82 express high, intermediate-high and low basal levels of Bcl-2, respectively, which are inversely correlated with the sensitivities of the cell lines to treatment with oligodeoxynucleotide 2009 and the chemotherapeutic agents alone. Moreover, differences were found in the responsiveness of the cell lines to treatment with combinations of oligodeoxynucleotide 2009 and the chemotherapeutic agents. In the cell lines NCI-H69 and SW2, all combinations resulted in synergistic cytotoxicity. In NCI-H69 cells, maximum synergy with a combination index of 0.2 was achieved with the combination of oligodeoxynucleotide 2009 and etoposide. In SW2 cells, the combination of oligodeoxynucleotide 2009 and doxorubicin was the most effective (combination index = 0.5). In the cell line NCI-H82, which expresses a low basal level of Bcl-2, most of the combinations were slightly antagonistic. Our data suggest the use of oligodeoxynucleotide 2009 in combination with chemotherapy for the treatment of small-cell lung cancer that overexpresses Bcl-2.

[Autoimmune hemolysis after Catergen ([+]-cyanidanol-3)]. / Autoimmunhämolyse nach Catergen ([+]-Cyanidanol-3).

Clinical and serological data in 5 cases of autoimmune hemolysis following therapy with Catergen are reported and compared to the data in similar literature reports. The main argument in favour of Catergen as causative agent in our 5 cases was rapid remission of hemolysis within 2 1/2 to 10 weeks of withdrawing Catergen treatment. Besides causing hemolysis mediated by drug dependent antibodies, long term treatment with Catergen may induce formation of IgG autoantibodies against red blood cells with or without overt hemolysis.

Immunotoxins recognising a new epitope on the neural cell adhesion molecule have potent cytotoxic effects against small cell lung cancer.

The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE). SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC. The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of [3H]leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively. Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR. Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE. In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill. SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins. SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE. These cells were still sensitive to SEN7-bR.

A comparison of 67Cu- and 131I-labelled forms of monoclonal antibodies SEN7 and SWA20 directed against small-cell lung cancer.

The intact anti-SCLC monoclonal antibody (MAb) SEN7 and its F(ab')2 were labelled with the beta-emitting isotope 67Cu. Both materials retained their biological activity in vitro as determined by the Lindmo assay. In a direct comparison of in vivo distribution in a xenograph model, 131I- and 67Cu-labelled intact SEN7 showed similar absolute tumour accumulation. Blood levels were markedly lower in the case of the 67Cu-labelled antibody, resulting in improved tumour:blood ratios which reached a maximum of 13:1 compared with only 4.5:1 for 131I-SEN7. In the case of the 67Cu-labelled F(ab')2, very high accumulation of the nuclide was observed in the kidney. Levels of radio copper in liver and spleen were also found to be significantly raised when compared with radio iodine. SWA20, a MAb which had previously failed to show any selective in vivo accumulation in tumour xenografts when labelled with radio iodine showed higher and more stable tumour accumulation when labelled with 67Cu.

In-process cell counts during preparation of platelet concentrates from buffy coats.

To ensure the quality of platelet concentrates (PCs), we studied in-process recoveries of blood cell counts in pooled PCs derived from four or five buffy coats (BCs) from Biopack Compoflex Systems in Bern (PC-BC/4 or PC-BC/5) and from five BCs from Optipac (Baxter) in Zurich (PC-BC/5). BCs were pooled employing a sterile connecting device and flushing them with 300 mL of platelet additive solution. The pools were centrifuged for 12 min at 500 g at 20 degrees C and filtered with PALL's Auto-Stop BC-leukocyte removal filter. Automated platelet counting was performed on whole blood donation, on single BC, on pooled BC and in the final product. Four out of 10 PC-BC/4 (= 40%) and 29 out of 30 PC-BC/5 (= 97%) had a total platelet count of > 200 x 10(9) platelets. Average percentage recoveries in PC compared to the pre-centrifugation BC pools were similar with the Biopack Compoflex and the Optipac systems, 62% and 57% respectively, whereby the absolute platelet count per one donation was similar, i.e. 49.5 x 10(9), 55 x 10(9) and 53 x 10(9) in PC-BC/4 and PC-BC/5 from Bern and PC-BC/5 from Zurich. There was a significant positive correlation between the inital number of BCs taken for pooling and the final platelet counts in the PCs. In order to recover a minimal platelet content of 200 x 10(9) platelets per pooled unit, it is safer to start out with five rather than with four donations unless recoveries during the production steps can be improved.