Human monoclonal antibody against a tissue polypeptide antigen-related protein from a patient with a signet-ring cell carcinoma of the stomach.

For a comparative study of the humoral immunity of patients with gastric signet-ring cell carcinoma, lymphocytes from spleen and lymph nodes were fused with the heteromyeloma SPM4-0. Immunoglobulin-producing clones were primarily tested in binding assays on autologous and allogeneic tumor cells and tissues. One of the resulting human monoclonal antibodies, designated 56/16 (IgM, lambda), was found to be suitable for a detailed biochemical characterization. Immunoblotting and comparative two-dimensional gel electrophoresis on cell and tissue extracts as well as on preparations of the cytoskeleton revealed that the main epitope is not an integral membrane molecule but a degradation product of cytokeratin 8, which is a main component of the tumor marker, tissue polypeptide antigen. The Mr 38,000/45,000 antigen could be identified in tumor and normal tissues, with highest expression in secretory cells and organs. Thus, the human monoclonal antibody 56/16 might represent an immune response in the patient against breakdown products of cytokeratin 8, which are released from the tumor cells during cell division, secretion, or cell death. A possible association of the antibody with the secretory activity of signet-ring carcinoma cells is discussed.

SC-1, a functional human monoclonal antibody against autologous stomach carcinoma cells.

Human monoclonal antibodies were isolated from stomach carcinoma patients by the fusion of spleen and lymph node lymphocytes with the heteromyeloma line SPM4-0. Initial screening was carried out on autologous primary tumor cell cultures in an adhesion assay in order to select surface-reactive functional antibodies. This was followed by live cell immunoperoxidase staining assays. The human monoclonal antibody SC-1 was found to selectively react with cultured cells isolated from autologous and allogeneic stomach carcinoma patients, and with cryostat sections of the primary tumors. More extensive screening revealed that the antibody showed no reactivity with a wide range of tumor tissues and normal cells. Some reactivity was present on fetal tissues. SC-1 inhibits movement of the autologous tumor cells and identifies a protein with a molecular weight of 50,000. This study demonstrates that, with the use of selective screening assays on primary tumor material, it is possible to isolate antibodies which not only result from an immune response in the patient but also interfere with intercellular processes of tumor cells.

Differential expression of CD44 splice variants in intestinal- and diffuse-type human gastric carcinomas and normal gastric mucosa.

Immunohistochemical screening of gastric adenocarcinomas from 42 different patients revealed variant CD44 expression in all specimens tested. Adenocarcinomas of the intestinal type were strongly positive for epitopes encoded by variant exons v5 and v6, whereas diffuse-type adenocarcinomas predominantly expressed only exon v5. Normal stomach mucosa was stained by an exon v5-specific monoclonal antibody within the foveolar proliferation zone and on mucoid surface epithelium. Areas of intestinal metaplasia reacted positively with monoclonal antibodies specific for exons v5 and v6. Analysis of RNA expression revealed dramatic differences between normal mucosa and adenocarcinomas. Whereas in normal epithelium only two CD44 variant RNAs containing exons v5 and/or v6 could be detected, intestinal-type tumors yielded a much more complex pattern of amplification products which hybridized to exons v5 and v6. A similar complex expression pattern of CD44 variants was observed in three cell lines established from intestinal-type tumors. In a sample of a diffuse-type tumor, expression of exon v5, but not v6, could be detected, confirming the data obtained with immunohistochemistry. These differences in variant exon v6 expression observed between diffuse-type and intestinal-type stomach adenocarcinomas establish variant CD44-specific antibodies as a tool in gastric cancer diagnosis and also support the theory of different origins for these tumor types.

Characterization of glycosylphosphatidylinositol-linked molecule CD55/decay-accelerating factor as the receptor for antibody SC-1-induced apoptosis.

The human monoclonal antibody SC-1 induces apoptosis of stomach carcinoma cells and is currently used in a clinical Phase II trial. The antibody binds to a target molecule that is preferentially expressed on diffuse- and intestinal-type stomach cancer cells and shows a very restricted expression on other normal and malignant tissues. In this paper, we show that the SC-1 receptor is a stomach carcinoma-associated isoform of CD55 [membrane-bound decay-accelerating factor (DAF)-B] with a relative molecular mass of approximately 82 kDa. The antigenic site of SC-1 is an N-linked carbohydrate residue. Cross-linking of the DAF receptor increases apoptotic activity. SC-1 binding induces tyrosine phosphorylation of three proteins of approximately 60, 75, and 110 kDa, whereas a serine residue of an approximately 35-kDa protein is dephosphorylated. Expression of caspase-3 (CPP32) and caspase-8 (FLICE) is elevated, and activation of these caspases occurs. These data show that a tumor-specific variant form DAF is involved in apoptosis and can be used for adjuvant therapeutical purposes on gastric carcinoma.

The "early birds": natural IgM antibodies and immune surveillance.

Precancerous epithelial lesions are sites of uncontrolled cellular proliferation generated by irreversible genetic alterations. Not all of those lesions progress to invasive cancer, some may even regress, but the early detection of abnormal cells can be crucial for patient survival. Immune surveillance mechanisms are responsible for the removal of transformed cells and antibodies play an important role in these immune processes. In the past, analysis of the immunoglobuline repertoire has focused mainly on xenoimmunizations or the investigation of cancer patient immunity. The human hybridoma technology (Trioma technique) offers the unique possibility to study the humoral immunity of healthy people. Using this technique a series of tumor-binding antibodies could be isolated which all have several features in common: they are germ-line coded IgM antibodies, they predominantly bind to carbohydrates on post-transcriptionally modified antigens, they induce apoptosis and, most importantly, they detect not only malignant cells but also precursor stages. These data demonstrate that the body has a comprehensive defense system against malignant cells based on the production of natural antibodies.

Importance of different CD44v6 expression in human gastric intestinal and diffuse type cancers for metastatic lymphogenic spreading.

In 42 human gastric adenocarcinomas of intestinal (n = 25) and diffuse types (n = 17) the expression of CD44v6 splice variants was investigated immunohistochemically and compared with the pattern of lymphogenic tumor spreading. Distinct differences were observed between the two cancer types: 92% of intestinal-type tumors expressed CD44v6 as in the intestinal metaplasia in chronic atrophic gastritis, while v6 expression occurred in only 17% of diffuse-type cancers. The analysis of RNA expression confirmed the immunohistochemical data. Intestinal-type cancers yielded a much more complex pattern of amplification products hybridizing to exon v6 than did normal mucosa, whereas diffuse-type tumors did not express exon v6. Also the pattern of lymphogenic spreading was quite different between the two cancer types: in diffuse-type tumors only a sinus carcinosis without CD44v6 expression was observed in a significantly higher number of lymph nodes than in intestinal-type cancers, which showed in particular infiltrative lymph node metastases always with CD44v6 expression as in the primary tumors. When infiltrative lymph node invasion occurred in v6-negative diffuse-type cancers, v6 neoexpression was also demonstrable in the lymph node metastases. Additionally, the number of infiltrative lymphogenic metastases increased with more extensive v6 expression in primary gastric cancers of both types. These data suggest that the expression of CD44v6 isoform is important for the infiltrative spreading of tumor cells into lymph nodes. Additionally, the phenotypic similarities in v6 expression between intestinal metaplasia and intestinal-type cancers, but not of tumors of diffuse type, may support the Correa hypothesis.

Death by stress: natural IgM-induced apoptosis.

Stress kills and hence should be avoided. On the other hand, stress induction can be used to remove malignant cells by inducing cellular suicide. Natural IgM antibodies act as first-line defense in immune surveillance. These antibodies selectively kill aberrant cells by using different apoptotic stress mechanisms. They can be isolated from patients but also from healthy donors by using the human hybridoma technology. They are components of the innate immunity, and, on the basis of specific screening methods, should also be detectable in any other individual. The three tumor-specific, apoptosis-inducing natural IgM antibodies described in this review are good examples for stress-induced apoptosis and nature's resourceful ways to fight malignant growth.

Monoclonal antibodies which prevent experimental lung metastases. Interference with the adhesion of tumour cells to laminin.

Cellular adhesion is important during metastasis, as metastatic cells must escape from the primary site into lymph and blood systems, there to adhere specifically to sites in distant organs. We have recently selected monoclonal antibodies which prevent adherence of B16 mouse melanoma cells to tissue culture dishes, and also markedly reduce experimental lung metastasis in mice when injected before or with the tumor cells. Here, we investigated which step in the metastatic process may be affected by the antibodies. The possible inhibitory effect of antibody on tumour cell adherence to vascular endothelial monolayers and to purified components of the underlying extracellular matrix - fibronectin, laminin and collagen type IV - was studied using in vitro assays. We found that the antibodies significantly blocked attachment to laminin, suggesting that specific basement membrane components play an important role in attracting or otherwise modifying the behaviour of metastatic tumour cells.

Natural IgM antibodies, the ignored weapons in tumour immunity.

During its lifetime each multi-cellular organism is permanently exposed to infectious agents and transformed cells. Without an early recognition and a rapid elimination system, there would be no development and no life. The innate or natural immunity, seems to be more important for the detection of "foreign" cells and particles than has been thought. Even if not every transformed cell has the ability and potency for malignant behaviour, the important question is not, why malignant cells arise, but instead, why malignancy occurs so infrequently. We have shown in a recent paper, by using the human hybridoma technology, that tumour immunity is not induced by malignant cells, but instead the result of innate immunity and that natural IgM antibodies play an important role in immunosurveillance mechanisms against transformed cells in humans (Brändlein et al., 2003b). In this review typical features of natural IgM antibodies are discussed and tumour-specific reactivities and different apoptotic functions on epithelial cancer cells are illustrated.

Immortalized B-lymphocytes from rheumatoid synovial tissue show specificity for bacterial HSP 60.

Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 or Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process.

Tumor-specific apoptosis induced by the human monoclonal antibody SC-1: a new therapeutical approach for stomach cancer.

Stomach cancer is one of the most frequently occurring cancers worldwide, with a very poor prognosis, even after complete gastrectomy. We describe here an alternative therapeutical approach using a human monoclonal antibody (SC-1), which was isolated from a patient with diffuse-type gastric adenocarcinoma. We demonstrate that the antibody significantly reduces stomach cancer growth in vivo, by inducing tumor-specific apoptosis and that the antibody, even delivered in high doses, shows no toxic crossreactivity to other organs or tissues. The data presented here show that tumor-specific apoptosis can be induced and they give rise to the hope that human monoclonal antibodies with biological activity might present a completely new type of adjuvant cancer therapy.

CFR-1 receptor as target for tumor-specific apoptosis induced by the natural human monoclonal antibody PAM-1.

The human monoclonal antibody PAM-1 was isolated from a patient with stomach cancer. The germ-line coded IgM antibody identifies a recently described 130 kDa variant of CFR-1 (cysteine-rich fibroblast growth factor receptor 1). This CFR-1/PAM-1 receptor is post-transcriptionally modified and over-expressed on human epithelial tumors and carcinoma pre-cancer lesions such as H. pylori induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung and cervical intraepithelial neoplasia. Furthermore, the expression of CFR-1/PAM-1 correlates with the proliferation rate and increases with the grade of malignancy. This study demonstrates that the human monoclonal antibody PAM-1 inhibits cell growth and induces apoptosis, in vitro and in vivo. Both, the unique tumor-specific expression of the CFR-1/PAM-1 receptor and the growth inhibitory effect of the PAM-1 antibody makes this combination a good diagnostic and therapeutic tool for all kinds of epithelial cancers and precursor lesions.

Adjuvant therapy for gastric adenocarcinoma with the apoptosis-inducing human monoclonal antibody SC-1: first clinical and histopathological results.

In a first clinical trial with the apoptosis-inducing human antibody SC-1 eight patients with poorly differentiated stomach adenocarcinoma of diffuse-type received 20 or 30 mg of purified SC-1 antibody intravenously, followed 24 or 48 h later by gastrectomy and lymphadenectomy. In seven cases a significant induction of apoptotic activity was measured in primary tumors as compared with earlier biopsy material and in five patients a significant regression of tumor mass could be determined histopathologically. No toxic crossreactivity was observed with normal tissue or organs of patients.

Deactivation of regulatory proteins hnRNP A1 and A2 during SC-1 induced apoptosis.

Phosphorylation and activation of caspases play an important role in the induction of apoptosis. During tumor specific apoptosis, induced by the human monoclonal antibody SC-1, tyrosine phosphorylation and serine dephosphorylation of several proteins is observed. In this paper we describe the identification of two dephosphorylated proteins as heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNP A1, hnRNP A2). The dephosphorylation of these proteins is important for apoptosis since the amount of apoptotic cell death can be decreased by the specific serine/threonine phosphatase inhibitor okadaic acid. We also investigated the effect of serine kinase inhibitor H7 on SC-1 induced apoptosis, which leads to a dose dependent increase in apoptosis. We could also show that 24 hours after the induction of apoptosis the hnRNP A1 protein is cleaved into different cleavage products. Further, we found a decreased expression of caspase-2 in early apoptosis signalling and an overexpression 24 hours after induction of apoptosis. Our results show that the phosphorylation status of the hnRNP A1 and A2 plays a significant role in early SC-1 induced apoptosis signalling and further indicate the role of caspase activation during the apoptotic process.

Electro-immortalization of B lymphocytes isolated from stomach carcinoma biopsy material.

The advantages of electrofusion were used to immortalize the small number of B-cells from fresh biopsy material taken from a gastric carcinoma of a patient. Two stable human antibody secreting clones could be produced which exhibited functional activity against the autologous tumour cells (inhibition of cell adhesion and immunofluorescence staining of the membranes). This shows that a variety of hitherto inaccessible B lymphocyte populations from other human organ biopsies can be immortalised by the improved electrofusion technique.

Natural IgM antibodies: from parias to parvenus.

Over the years, natural IgM antibodies were considered as the parias among the immune competent molecules. Their characteristic properties, like low affinity, cross-reactivity and pentameric structure, were assessed as difficult and nebulous. Today, mainly based on the persistent work of a few researchers and the key discoveries on innate immunity, natural IgM antibodies are "back on stage". Their important role in the immune response against invasive particles, modified self-components and altered cells is accepted. All the so far negatively judged features have to be seen in a different light, e.g. low affinity seems to be good for function and does not exclude specificity, cross-reactivity is no longer judged as unspecific, but instead as a very economic way of immune recognition and the pentameric structure is important for binding capacity and functional activities. In addition, with the use of natural IgM antibodies, a new field of tumour-specific targets has been encountered, the carbo-neo-epitopes, which are commonly found on post-transscriptionally modified membrane receptors. Having understood the typical features of natural IgM antibodies, their renaissance opens a new area of cancer therapeutics and diagnostics.

Monoclonal antibodies that prevent adhesion of B 16 melanoma cells and reduce metastases in mice: crossreaction with human tumor cells.

Monoclonal antibodies raised against B 16 melanoma cells in syngeneic mice were functionally screened for their ability to inhibit cell adhesion in tissue culture. Three of these antibodies (16/43, 16/77, 16/82), when preinjected into C57BL/6 mice, markedly reduced the number of experimental lung metastases produced by B 16 cells, possibly by interference with their adhesion to the lung endothelia. We now report that these monoclonal antibodies block in vitro attachment of the majority of human melanoma cell lines tested and also of carcinoma, neuroblastoma, and glioblastoma cells from both mice and humans but untransformed cell lines such as 3T3 mouse or MRC-5 human fibroblasts are not affected. The antibodies also react with mouse teratocarcinoma stem cells (F9, PCC4) but not with differentiated teratocarcinoma lines (PYS-2, 944). Furthermore, the antiadhesion activity of the antibodies could be quantitatively absorbed by intact human and mouse tumor cells but not by untransformed cells, suggesting that the corresponding antigens may represent tumor-associated cell surface components. Correspondingly, the antigens were found on simian virus 40-transformed 3T3 mouse fibroblasts and are expressed in a temperature-sensitive fashion in chicken fibroblasts transformed with a temperature-sensitive Rous sarcoma virus. On "immunoblots" of NaDodSO4-containing gels the three selected antibodies (16/43, 16/82, 19/1) were absorbed by antigens with molecular weights of 40,000 and 50,000.

Monoclonal antibodies inhibit the adhesion of mouse B 16 melanoma cells in vitro and block lung metastasis in vivo.

Seven monoclonal antibodies against mouse B 16 melanoma cells (produced in syngeneic C57BL/6 mice) were selected that blocked the adhesion of melanoma cells to tissue culture dishes. These antibodies were found to be directed against antigens on the surface of mouse B 16 melanoma cells but not on normal mouse cells such as 3T3 fibroblasts. Similarly, the antigens could not be detected in normal mouse tissues (e.g., lung, kidney, liver) but were found in lungs colonized by B 16 melanoma cells. Significantly, three of these antibodies virtually abolished lung colonization of highly invasive B 16 sublines injected into the animals' bloodstream. They exerted their effect both when preabsorbed by the melanoma cell in vitro and when delivered to the animals prior to the tumor cells. It is suggested that monoclonal antibodies might be a promising tool for preventing metastasis.

Control of cell locomotion: perturbation with an antibody directed against specific glycoproteins.

A murine monoclonal antibody, SLOW-1, was selected, which inhibits the locomotion of chick embryo fibroblasts (the immunizing cells) in tissue culture. The antibody, an IgM, cross-reacts in locomotion assays with a number of tumor and untransformed cells, and on fixed and permeabilized cells binds 1-5 X 10(5) target sites with an affinity of 10(-8) M. The antigen can be extracted from cells with isotonic buffers containing EGTA, binds to Concanavalin A, and when analyzed on SDS gels by immunoblotting, two major antigenic glycoproteins are detected at 57 kd (isoelectric point, 5.1) and at 44 kd (isoelectric point, 5.4). The antigenic site involves galactosyl or mannosyl residues, or both, within a complex, N-linked carbohydrate tree. The possible contribution of the SLOW-1 antigen to a common control system of locomotion operating over the cell surface is discussed.

Monoclonal antibodies NORM-1 and NORM-2 induce more normal behavior of tumor cells in vitro and reduce tumor growth in vivo.

In this study, large numbers of hybridomas (produced by syngeneic immunization with B16 mouse melanoma and fusion with NS-1 myeloma cells) were screened for the production of antibodies that affected morphology and growth of animal and human tumor cells in vitro. Two such antibodies, NORM-1 and NORM-2 (both IgG2a), inhibited the growth of B16 melanoma cells in soft agar and increased the serum requirements of tumor cells in tissue culture. Antibody NORM-2 also inhibited the growth of SV40-transformed 3T3 cells in agar and caused them to deposit more fibronectin into extracellular matrix. These antibodies thus seem to induce a more normal behavior of tumor cells in vitro. In vivo both antibodies reduced the number of growing lung tumors of B16 melanoma in C57BL/6 mice by 70%-90% when injected 3 days after the tumor cells. By immunoprecipitation of 35S-methionine-labeled cell extracts, NORM-2 antibody recognized a 59 kd protein in B16 mouse and in A375 human melanoma cells but not in 3T3 fibroblasts.