Diagnostic and therapeutic potential of a human antibody cloned from a cancer patient that binds to a tumor-specific variant of transcription factor TAF15.

Human hybridoma technologies permit the cloning of patient antibodies that may have desirable qualities. In this study, we report the isolation of a natural IgG antibody from a stomach cancer patient that illustrates novel diagnostic and therapeutic uses. Human antibody PAT-BA4 recognizes a tumor-specific variant of the transcription factor TATA-binding protein-associated factor 15 (TAF15) that is expressed on the plasma membrane of stomach cancer and melanoma cells but not healthy tissues. TAF15 is a member of the multifunctional TET protein family involved in mRNA transcription, splicing, and transport that is normally expressed only in the cytoplasm and nucleus of fetal or adult tissue cells. However, in malignant cells, TET family members including TAF15 seem to be involved in cell adhesion and spreading. In support of this likelihood, we found that PAT-BA4 inhibited tumor cell motility and tumor cell adhesion. Our findings define a role for a tumor-specific TAF15 antigen in malignant processes.

Natural antibodies and cancer.

The innate or natural immunity is the basis and key for all immune processes. Specific receptors on macrophages, dendrites, NK cells and natural antibodies producing B cells act as a first line defense and remove all 'foreign' and potentially harmful substances, that is, bacteria, viruses, cellular waste, modified molecules and, most importantly, cancer cells. Recognition and removal of transformed cells is a lifelong task of immune surveillance processes. Antibodies are hallmark components of this anti-cancer activity. To investigate their nature, specificity, and function, we used the human hybridoma technology for isolating antibodies from cancer patients. These were then tested with a panel of assays against cancer cell lines in vitro and in vivo. Interestingly, all the tumor-specific antibodies we found were germ-line coded and belonged nearly exclusively to the IgM class. Furthermore, they all bound to new carbohydrates on post-translationally modified cell surface receptors on malignant cells. So far no affinity maturated immunoglobulins detecting tumor-specific peptides were found. However, only the presentation of peptide motifs can create an immunological memory. In general malignant cells are detected at very early precursor stages and manifest tumors can be considered as exceptional events. In addition, malignant cells are neither infectious nor hide intracellularly like viruses and some bacteria. Therefore, it makes sense that anti-tumor immunity seems to be solely a part of the natural immunity and a memory is not needed and therefore not induced. This indicates that the tumor immunity seems to be restricted to innate immune mechanisms and the instruments used by nature, like natural antibodies, are obviously excellent therapeutics.

High level expression of functional human IgMs in human PER.C6 cells.

Natural IgM antibodies play an important role in the body's defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6 cells-a human cell line-to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6 cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.

Natural human immunoglobulins in cancer immunotherapy.

The natural or innate immunity is the first-line defense against transformed cells. It guarantees the recognition and removal of malignant cells at an early stage and makes manifest cancers an exceptional event. Natural antibodies, which are predominantly IgM molecules, play a major role in these defense mechanisms and they have some typical features in common. They are coded by specific germline families and equipped mainly with lambda-chains, in contrast to the majority of circulating antibodies. The targets that are recognized by these antibodies are not newly synthesized proteins, but instead post-translationally modified carbohydrate structures on membrane-bound glycoproteins and glycolipids. Another typical feature of these natural IgM antibodies is their ability to induce apoptosis in vitro and in vivo in a death domain-independent manner. These results show that natural IgM antibodies represent a huge reservoir of therapeutic antibodies.

A new tumor-specific variant of GRP78 as target for antibody-based therapy.

The chaperone GRP78 is a member of the heat-shock protein 70 (HSP70) family and is responsible for cellular homeostasis by preventing stress-induced apoptosis. GRP78 is expressed in all cells of the body. In malignant cells, which are permanently exposed to environmental stress, GRP78 is overexpressed and increased levels can be found in the cytoplasm and on the cell membrane. Thus, GRP78 promotes tumor proliferation, survival, metastases and resistance to a wide variety of therapies. Like other tumor-specific membrane molecules, GRP78 can also be present on cancer cells in a variant form. This modification qualifies it as a target for immune surveillance and antibody responses. The fully human monoclonal IgM antibody, SAM-6, was isolated from a gastric cancer patient and it binds to a new variant of GRP78 with a molecular weight of 82 kDa. The epitope is an O-linked carbohydrate moiety and is specific for malignant cells. These data show that cancer-specific modifications of cell-surface protection molecules are (a) subject of an immune response and (b) ideal targets for new therapeutical approaches.

Neoadjuvant therapy of gastric cancer with the human monoclonal IgM antibody SC-1: impact on the immune system.

Adjuvant therapies for minimal residual disease are a promising approach to improve the poor survival rates after surgery of gastric tumors. A pilot study of a neoadjuvant therapy was performed using a human monoclonal IgM antibody (SC-1) specifically inducing apoptosis in signet ring cell stomach carcinomas. However, scarce information exists on how such a treatment affects the immune system, in particular what are the effects of apoptosis induction and infusion of large amounts of IgM. Thus, the leukocyte composition (CD3, CD4, CD8, CD19, CD16+56, CD14) and several cytokines (TNF-alpha, IL6, IL12, IFN-gamma, GM-CSF, Neopterin) before and after SC-1 application were measured and compared to results of patients that underwent surgical removal of gastric carcinoma without antibody treatment. After SC-1 application, an increase in TNF-alpha and a decrease of lymphocytes and CD3+ T-cells but in the range obtained in healthy individuals was observed before surgery. After surgery, the IL6 levels increased and the TNF-alpha levels remained at the elevated level. Furthermore, there was a significant drop in lymphocytes and CD3+ T-cells. These effects were due to the surgical treatment. Other parameters did not show significant changes. It seems that the application of an apoptosis-inducing antibody prior to surgery of gastric tumors has mild if any effect on the immune system. Therefore, from an immunological point of view, the treatment with this monoclonal antibody is extremely safe.

Tumors: too sweet to remember?

Immunity, based on a natural and an educated system, is responsible for recognition and elimination of infectious particles, cellular waste, modified self and transformed cells. This dual system guarantees that dangerous particles are removed immediately after appearance and that a memory with maturated weapons exists, if the organism is re-infected by the same particle. For malignant cells, however, the immune response seems to be restricted to innate immunity, because at least for the humoral response, all so far detected tumor-specific antibodies belong to the natural immunity. In this review we try to explain why malignant cells might be "too sweet" to induce a memory.

Natural antibodies and cancer.

Immunity is not only responsible for recognition and elimination of infectious particles, but also for removal of cellular waste, modified self structures and transformed cells. Innate or natural immunity acts as a first line defense and is also the link to acquired immunity and memory. A striking phenomenon of immunity against malignant cells is that neither in animals nor in humans affinity-maturated tumor-specific IgG antibodies have been detected so far. All tumor-specific isolated antibodies were germ-line coded natural IgM antibodies. It's also a fact that these IgM's preferentially bind to carbohydrate epitopes on post-transcriptionally modified surface receptors and that they all induce a cancer-specific apoptosis, by triggering the intrinsic apoptotic pathway. From an evolutionary point of view, this makes sense because cancer cells are not infectious, so there is no need for memory. Natural IgMs bind to conservative structures because they are coded by a limited set of genes and they use apoptosis, the "clean" way of killing, to avoid inflammatory processes.

The human IgM antibody SAM-6 induces tumor-specific apoptosis with oxidized low-density lipoprotein.

Lipids are essential for normal and malignant cells during growth and differentiation. The turnover is strictly regulated because an uncontrolled uptake and accumulation is cytotoxic and can lead to lipoapoptosis: lipoptosis. The human monoclonal antibody SAM-6 binds to a cell surface receptor on malignant cells and to oxidized low-density lipoprotein (LDL). SAM-6 induces an excess of intracellular lipids, by overfeeding malignant cells with oxidized LDL, via a receptor-mediated endocytosis. The treated cells overaccumulate depots of cholesteryl esters and triglycerides. This lipid overaccumulation is tumor specific; nonmalignant cells neither bind the antibody nor harvest lipids after incubation. Because for both forms of apoptosis, the death domain dependent ("extrinsic") and independent ("intrinsic"), the activation of proteases is crucial, we also investigated this pathway in more detail. It was found that shortly after internalization of antibody/oxidized LDL/receptor complex and formation of lipid depots, cytochrome c is released by mitochondria. Followed by this, initiator caspase-8 and caspase-9 and effector caspase-3 and caspase-6 are activated. The mechanism of mitochondrial trigger (e.g., by free fatty acids) is under investigation. However, the present data indicate that the SAM-6 antibody induces an intrinsic-like form of apoptosis by overfeeding malignant cells with lipoproteins.

Natural IgM antibodies: the orphaned molecules in immune surveillance.

Natural IgM antibodies are typical victims of prejudices which originated in the mid 80 s. Over the years, these molecules were considered as the pariahs among the immune competent molecules and their characteristic properties, like low affinity, cross-reactivity and pentameric structure, were assessed as useless, difficult, nebulous, etc. Today, mainly based on a few scientists' persistent work and the key discoveries on innate immune recognition, natural IgM antibodies are "back on stage". Their role in the immune response against bacteria, viruses, fungi and possibly modified self-components as well as in therapy and diagnosis of malignancies is accepted. All the so far negatively judged features are seen in a different light, e.g. low affinity seems to be good for function and does not exclude specificity, and cross-reactivity is no longer judged as unspecific, but instead as a very economic way of immune recognition. And at last, with the use of natural IgM antibodies, a new field of tumor-specific targets has been encountered, the carbo-neo-epitopes. Therefore, by having learned from nature, the renaissance of natural IgM antibodies opens a new area of cancer therapeutics and diagnostics.

Human antibody SC-1 reduces disseminated tumor cells in nude mice with human gastric cancer.

Advanced gastric cancer is a systemic disease that requires adjuvant therapy targeted at eliminating disseminated tumor cells (DTCs). We investigated whether the apoptosis-inducing human monoclonal IgM antibody SC-1 was able to reduce the number of disseminated gastric cancer cells in blood and bone marrow. Human gastric tumor specimens with positive expression of the SC-1 receptor were transplanted in nude mice with metastasizing gastric cancer. After tumor growth (4-6 weeks) animals were randomly allocated to intraperitoneal 100 microg SC-1 (n=23) or 100 microg human IgM (n=23). One week later, animals were sacrificed and blood and bone marrow specimens were obtained. A nested RT-PCR for cytokeratin 20 (CK-20) from blood and bone marrow of mice was performed for detection of disseminated tumor cells. Animals receiving SC-1 had significantly fewer DTCs than did control animals (p=0.0011). None of the SC-1 mice had DTCs simultaneously in both blood and bone marrow versus four of the control animals (p=0.0363). The reduction of DTCs in SC-1 animals was due to reduction in bone marrow (p=0.032 compared to controls), but not in blood (p=0.1158). Treatment with SC-1 significantly reduced the number of DTCs in bone marrow in this animal model.

Lipoptosis: tumor-specific cell death by antibody-induced intracellular lipid accumulation.

A balanced lipid metabolism is crucial for all cells. Disturbance of this homeostasis by nonphysiological intracellular accumulation of fatty acids can result in apoptosis. This was proven in animal studies and was correlated to some human diseases, like lipotoxic cardiomyopathy. Some metabolic mechanisms of lipo-apoptosis were described, and some causes were discussed, but reagents, which directly induce lipo-apoptosis, have thus far not been identified. The human monoclonal IgM antibody SAM-6 was isolated from a stomach cancer patient by using the conventional human hybridoma technology (trioma technique). The addition of SAM-6 to tumor cells leads to an increase in the intracellular accumulation of neutral lipids, followed by tumor cell apoptosis. The antibody SAM-6 does not react with noncancerous human epithelial and fibroblastic cells, because the M(r) 140000 membrane molecule, recognized by the antibody, is specifically expressed on human malignant cells. The antibody is coded by the germ-line genes IgHV3-30.3*01 and IgLV3-1*01 and is a component of the innate immunity to cancer. In this article, we describe an antibody-induced tumor-specific cell death, named lipoptosis. This is, to our knowledge, the first description of this specific form of lipo-apoptosis as an antibody-mediated mechanism of tumor cell killing.

PAM-1, a natural human IgM antibody as new tool for detection of breast and prostate precursors.

Early detection and differential analysis of premalignant lesions are very important for both prognosis and therapy of cancer patients. A good source of diagnostic tools is the natural antibody pool of humans. Tumor-specific antibodies can be established by using hybridoma technology. The fully human germline-coded monoclonal IgM antibody PAM-1 was isolated from a patient with a stomach carcinoma. PAM-1 reacts with a post-transcriptionally modified isoform of membrane receptor CFR-1 which is overexpressed on almost all epithelial cancers of all types and origins. The expression of CFR-1/PAM-1 on precancerous stages of breast and prostate cancer was analyzed by immunohistochemistry and compared with normal breast and prostate tissue as well as adenocarcinomas of both. In addition FACS analysis was performed to detect receptor expression on benign and malign prostate cells. 73 different tissue samples of prostate and breast precancerous stages and prostate and breast carcinomas were analysed for CFR-1/PAM-1 expression immunohistochemically. The CFR-1/PAM-1 receptor was expressed on nearly all precancerous stages and carcinomas while normal breast and prostate tissue showed negative results. These results were confirmed by FACS analysis showing a CFR-1/PAM-1 expression only on prostate carcinoma cells but not on benign prostate hyperplasia cells. The unique expression of this new CFR-1/PAM-1 receptor makes the PAM-1 antibody an ideal diagnostic and even therapeutic tool for precancerous and cancerous epithelial lesions of the breast and the prostate.

Natural IgM antibodies and immunosurveillance mechanisms against epithelial cancer cells in humans.

Malignancy is like a chronic disease, and the immune system is permanently involved in recognizing and eliminating the transformed cells. The human hybridoma technology offers the unique opportunity to study the mechanisms, structures, and targets involved in recognition and elimination of aberrant cells. Thousands of tumor-reactive human monoclonal antibodies were isolated by this technique from cancer patients and from healthy donors, and all of these antibodies were IgM antibodies; no IgG and IgA antibodies were found. Fourteen of these antibodies were selected for DNA sequence analysis, characterization of their binding patterns, and determination of their origin and genetics. All of the IgM antibodies studied expressed only few or no mutations at all (germ-line coded), bound to carbohydrates on modified tumor-specific receptors and induced apoptosis. The degree of cross-reactivity to other tumors correlated reciprocally with the number of mutations in coding regions. By using an anti-idiotypic antibody we were able to show that the IgM-producing cells were of CD5+ B-cell origin. The data presented here indicate that the innate immunity and natural IgM antibodies play an important role in immunosurveillance mechanisms against epithelial tumors in humans.

Cysteine-rich fibroblast growth factor receptor 1, a new marker for precancerous epithelial lesions defined by the human monoclonal antibody PAM-1.

Precancerous epithelial lesions are sites of uncontrolled cellular proliferation, generated by irreversible genetic changes. Not all of these lesions progress to invasive cancer, some may even regress, but early detection of abnormal cells can be crucial for survival of the patient. Diagnosis is mainly performed by using morphological parameters. Proliferation markers can facilitate the analysis, if they show a consistent expression, and distinguish between healthy and malignant cells. The fully human monoclonal IgM antibody PAM-1 was isolated from a patient with stomach carcinoma and binds to a new variant of cysteine-rich fibroblast growth factor receptor 1 (CFR-1). This CFR-1/PAM-1 receptor is expressed on nearly all of the epithelial cancers of every type and origin, but not on healthy tissue. It is also present on precursor lesions found in: Helicobacter pylori-induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett's metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung, and cervical intraepithelial neoplasia. The unique, growth-dependent expression of this new CFR-1 isoform makes the PAM-1 antibody an ideal diagnostic tool for the detection of precancerous and cancerous lesions.

Human monoclonal IgM antibodies with apoptotic activity isolated from cancer patients.

Monoclonal antibodies are accepted as ideal adjuvant therapeutic reagents for all kinds of diseases. Polyvalent (cross-linking) and low-mutated IgM antibodies (less immunogenic) are believed to be the most effective weapons against cancer. The best sources for these types of antibodies are the cancer patients themselves. Using conventional hybridoma technology, not only are fully human monoclonal IgM antibodies isolated, but also new tumor-related targets can be identified using the same experimental approach. The resulting antibodies can be used directly for therapeutic purposes without further modulation and manipulation. This report describes five newly established human monoclonal IgM antibodies; antibody LM-1 that was isolated from a patient with lung cancer, antibodies PM-1 und PM-2 that were isolated from a patient with pancreatic cancer, and antibodies CM-1 and CM-2 which were isolated from a patient with colon carcinoma. The mainly germ-line encoded antibodies are specific for malignant tissues and show only restricted reactivity with healthy cells. When tested for in vitro functional activity, all five antibodies inhibit tumor cell proliferation of carcinoma cells by inducing apoptosis.

Nature's best weapons to fight cancer. Revival of human monoclonal IgM antibodies.

The unique features of monoclonal antibodies (specificity, effectiveness, purity and unlimited reproducibility) make them ideal tools for the specific treatment of all kind of diseases. The third generation of monoclonal antibodies for the treatment of human diseases will be, after murine and "humanised" murine immunoglobulins, fully human antibodies. The best source of human monoclonal antibodies are the antibody pools of cancer patients themselves with the best technique for generating them being conventional human hybridoma technology. This technique, will generate human monoclonal antibodies which will not only define important new targets on cancerous tissue, but will also provide the necessary therapeutic human antibodies in the fight against cancer.