EpCAM in carcinogenesis: the good, the bad or the ugly.

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is highly expressed on most carcinomas and therefore of potential use as a diagnostic and prognostic marker for a variety of carcinomas. Interestingly, EpCAM is explored as target in antibody-based therapies. Recently, EpCAM has been identified as an additional marker of cancer-initiating cells. In this review, we describe the controversial biological role of EpCAM with the focus on carcinogenesis: as an adhesion molecule, EpCAM mediates homophilic adhesion interactions, which in turn might prevent metastasis. On the other hand, EpCAM abrogates E-cadherin mediated cell-cell adhesion thereby promoting metastasis. Also, upon cleavage of EpCAM, the intracellular domain functions as a part of a transcriptional complex inducing c-myc and cyclin A and E. In line with these seemingly controversial roles, EpCAM overexpression has been associated with both decreased and increased survival of patients. Similarly, either induction or downregulation of EpCAM expression lowers the oncogenic potential depending on the cell type. As epigenetic dysregulation underlies aberrant EpCAM expression, we propose epigenetic editing as a novel approach to investigate the biological role of EpCAM, expanding the options for EpCAM as a therapeutic target in cancer.

Targeted delivery of a designed sTRAIL mutant results in superior apoptotic activity towards EGFR-positive tumor cells.

Previously, we have shown that epidermal growth factor receptor (EGFR)-selective delivery of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), by genetic fusion to antibody fragment scFv425, enhances the tumor-selective pro-apoptotic activity of sTRAIL. Insight into the respective contribution of the agonistic receptors TRAIL-R1 and TRAIL-R2 to TRAIL-induced apoptosis may provide a rational approach to further optimize TRAIL-based therapy. Recently, this issue has been investigated using sTRAIL mutants designed to selectively bind to either receptor. However, the relative contribution of the respective TRAIL receptors, in particular TRAIL-R1, in TRAIL signaling is still unresolved. Here, we fused scFv425 to designed sTRAIL mutant sTRAILmR1-5, reported to selectively activate TRAIL-R1, and investigated the therapeutic apoptotic activity of this novel fusion protein. EGFR-specific binding of scFv425:sTRAILmR1-5 potently induced apoptosis, which was superior to the apoptotic activity of scFv425:sTRAIL-wt and a nontargeted MOCK-scFv:sTRAILmR1-5. During cotreatment with cisplatin or the histone deacetylase inhibitor valproic acid, scFv425:sTRAILmR1-5 retained its superior pro-apoptotic activity compared to scFv425:sTRAIL-wt. However, in catching-type Enzyme-Linked ImmunoSorbent Assays with TRAIL-R1:Fc and TRAIL-R2:Fc, scFv425:sTRAILmR1-5 was found to not only bind to TRAIL-R1 but also to TRAIL-R2. Binding to TRAIL-R2 also had functional consequences because the apoptotic activity of scFv425:sTRAILmR1-5 was strongly inhibited by a TRAIL-R2 blocking monoclonal antibody. Moreover, scFv425:sTRAILmR1-5 retained apoptotic activity upon selective knockdown of TRAIL-R1 using small inhibitory RNA. Collectively, these data indicate that both agonistic TRAIL receptors are functionally involved in TRAIL signaling by scFv425:sTRAILmR1-5 in solid tumor cells. Moreover, the superior target cell-restricted apoptotic activity of scFv425:sTRAILmR1-5 indicates its therapeutic potential for EGFR-positive solid tumors.

Persistent downregulation of the pancarcinoma-associated epithelial cell adhesion molecule via active intranuclear methylation.

The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM-negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAM-positive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM-positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA-mediated silencing remained for 4 days, after which EpCAM re-expression increased in time, while M.SssI-mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.

EpCAM in morphogenesis.

Embryonic development is one of the most complex biological phenomena that involves the appropriate expression and synchronized interactions of a plethora of proteins, including cell adhesion molecules (CAMs). Many members of the diverse family of CAMs have been shown to be critically involved in the correct execution of embryonic development. The Epithelial Cell Adhesion Molecule (EpCAM) is an atypical cell adhesion molecule originally identified as a marker for carcinoma. However, recent insights have revealed that EpCAM participates in not only cell adhesion, but also in proliferation, migration and differentiation of cells. All of these processes are known to be fundamental for morphogenesis. Here, we review the current literature that establishes EpCAM as a protein involved in morphogenesis, starting from the earliest stages of embryogenesis and ending in organogenesis. In addition, we provide directions for further elucidation of the role of EpCAM in embryogenesis.

Viral chemokine-modulatory proteins: tools and targets.

The chemokine network is an extensive system that regulates many immune functions such as leukocyte locomotion, T cell differentiation, angiogenesis and mast cell degranulation. Tight control of chemokines is vital for proper immune function. Not surprisingly, viruses have found ways to subvert or exploit the immune system in order to persist in co-existence with their hosts. Several viral immune evasion genes encode proteins that modulate the chemokine network. We attempt to identify which aspects of the chemokine control mechanisms are susceptible to modulation. Chemokine-glycosaminoglycan interaction, extracellular processing of chemokines and chemokine scavenging will be discussed in the light of poxvirus and herpesvirus immune evasion. Viral chemokine-modulatory proteins may either be targets for anti-viral therapy or lead the way to new anti-inflammatory chemokine-modulating drugs.

Target cell-restricted apoptosis induction of acute leukemic T cells by a recombinant tumor necrosis factor-related apoptosis-inducing ligand fusion protein with specificity for human CD7.

Current treatment of human T-cell leukemia and lymphoma is predominantly limited to conventional cytotoxic therapy and is associated with limited therapeutic response and significant morbidity. Therefore, more potent and leukemia-specific therapies with favorable toxicity profiles are urgently needed. Here, we report on the construction of a novel therapeutic fusion protein, scFvCD7:sTRAIL, designed to induce target antigen-restricted apoptosis in human T-cell tumors. ScFvCD7:sTRAIL consists of the death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7. Treatment with scFvCD7:sTRAIL induced potent CD7-restricted apoptosis in a series of malignant T-cell lines, whereas normal resting leukocytes, activated T cells, and vascular endothelial cells (human umbilical vein endothelial cells) showed no detectable apoptosis. The apoptosis-inducing activity of scFvCD7:sTRAIL was stronger than that of the immunotoxin scFvCD7:ETA. In mixed culture experiments with CD7-positive and CD7-negative tumor cells, scFvCD7:sTRAIL induced very potent bystander apoptosis of CD7-negative tumor cells. In vitro treatment of blood cells freshly derived from T-acute lymphoblastic leukemia patients resulted in marked apoptosis of the malignant T cells that was strongly augmented by vincristin. In conclusion, scFvCD7:sTRAIL is a novel recombinant protein causing restricted apoptosis in human leukemic T cells with low toxicity for normal human blood and endothelial cells.

The human cytomegalovirus-encoded receptor US28 increases the activity of the major immediate-early promoter/enhancer.

The activation of the major immediate-early promoter (MIEP) is a key event in the cytomegalovirus replication cycle and is dependent on cellular transcription factors which are partially activated by viral proteins. Expression of the viral chemokine receptor homolog US28 results in constitutive activation of pro-inflammatory transcription factors that may be involved in the activation of the major immediate-early promoter/enhancer. Using reporter gene assays in human embryonic kidney cells, we found that US28 signaling was responsible for increased major immediate-early promoter/enhancer activity which was independent of beta-chemokine binding. Inhibition of nuclear factor-kappaB (NF-kappaB) only partially blocked the effect of US28, whereas treatment with a specific p38 mitogen activated kinase (MAPK) inhibitor fully abrogated the US28-induced enhancement of promoter activity. Our results suggest that during human cytomegalovirus (HCMV) infection, US28 in epithelial cells transactivates the major immediate-early promoter/enhancer via the activation of p38 MAPK and downstream signaling that partially involves NF-kappaB.

The modulation of angiogenesis in the foreign body response by the poxviral protein M-T7.

The foreign body response is characterized by enhanced recruitment of inflammatory cells. As the directional movement of cells is controlled by chemokines, disruption of the chemokine network would be an attractive approach to improve biocompatibility of an implanted material. The sequestration of chemokines by cell surface-expressed glycosaminoglycans (GAGs) is vital for in vivo chemokine activity. The myxoma virus encodes a soluble protein, M-T7, that interacts with conserved GAG-binding domains of chemokines to block chemokine-mediated leukocyte recruitment. We hypothesized that M-T7 might also affect the function of other inflammation-associated proteins in addition to chemokines that bind to GAG. In our studies, we focussed on the modulation of the GAG-binding molecules macrophage chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor-164 (VEGF164) in the inflammatory reaction against subcutaneously implanted degradable cross-linked dermal sheep collagen discs in AO rats. Genetic delivery of M-T7 delays the influx of macrophages into the collagen discs. In addition, angiogenesis around the implanted material was reduced. The discs revealed reduced levels of rat MCP-1 and rat VEGF164. This was not due to down regulation of transcription of the genes that encode MCP-1 and VEGF164. Our in vivo observations suggest that, in addition to chemokines such as MCP-1, M-T7 neutralizes VEGF164.

Use of the EGP-2/Ep-CAM promoter for targeted expression of heterologous genes in carcinoma derived cell lines.

EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated and assayed for their ability to control the expression of the EGP-2 cDNA, the green fluorescent protein, the luciferase reporter gene and the thymidine kinase and cytosine deaminase suicide genes. Expression of these chimeric constructs as assessed in a range of different cell lines was restricted to cell lines expressing EGP-2. In addition, only cells expressing EGP-2 were sensitive for gancyclovir after being transiently transfected with EGP-2 promoter-driven thymidine kinase. Deletion analyses defined 687 bp upstream as the basic proximal promoter region, which could confer epithelial-specific expression to the GFP reporter gene in vitro. As these EGP-2 sequences can confer promoter activity to reporter and suicide genes in an EGP-2 restricted manner, they may be useful for gene therapy of EGP-2 expressing carcinomas.

Specific immune responses against airway epithelial cells in a transgenic mouse-trachea transplantation model for obliterative airway disease.

BACKGROUND: Immune injury to airway epithelium is suggested to play a central role in the pathogenesis of obliterative bronchiolitis (OB) after clinical lung transplantation. In several studies, a rejection model of murine trachea transplants is used, resulting in obliterative airway disease (OAD) with similarities to human OB. To focus on the role of an immune response specifically against airway epithelium, we transplanted tracheas from transgenic mice expressing human epithelial glycoprotein (hEGP) on epithelial cells. We hypothesized that the immune response against the hEGP-2 antigen would result in OAD in the trachea transplants. METHODS: Tracheas from hEGP-2 transgenic and control nontransgenic FVB/N mice were heterotopically transplanted into FVB/N mice and harvested at week 1, 3, 6, and 9. Anti-hEGP-2 antibodies were determined in the recipient blood. The trachea grafts were analyzed for cellular infiltration, epithelial cell injury, and luminal obliteration. RESULTS: Recipients of transgenic tracheal grafts gradually developed anti-hEGP-2 antibodies. In the transgenic grafts, the submucosa was infiltrated predominantly by CD4+ T cells. Epithelial cells remained present but showed progressive abnormality. The tracheal lumen showed a mild degree of obliteration. All these changes were absent in nontransgenic FVB/N trachea transplants. CONCLUSION: The hEGP-2 antigen on the epithelial cells of transgenic trachea transplants induces specific humoral and cellular immune responses, leading to a mild form of OAD. It provides a suitable model for further investigation of the role of epithelial cells in the development of OAD in animals and OB in human-lung transplantation.

Detection of alloreactive T cells by flow cytometry: a new test compared with limiting dilution assay.

BACKGROUND: Frequencies of alloreactive T cells determined by limiting dilution assays (LDA) may not adequately reflect the donor-reactive immune status in transplant recipients. To reevaluate LDA frequencies, we developed a flow cytometry test for direct determination of alloreactive T-cell frequencies and compared these frequencies with classical LDA estimates of frequencies. METHODS: For determination of frequencies by flow cytometry, peripheral blood lymphocytes (or lymphocytes taken from primary mixed lymphocyte culture) were stimulated with either Epstein-Barr virus-transformed lymphoblastoid cell lines or T cell-depleted spleen cells and stained for intracellular interferon (IFN)-gamma production and CD69. In lung transplant recipients, frequencies of IFN+ alloreactive T cells were compared with LDA frequencies, that is, cytotoxic T lymphocyte precursors and helper T lymphocyte precursors. RESULTS: With flow cytometry, alloreactive T cells were detected after overnight allostimulation as IFN-gamma CD69bright cells (range, 0.1-0.58% and 0.1-0.66% of total CD4 and CD8 cells, respectively). Frequencies increased 25-fold or more when lymphocytes were prestimulated in primary mixed lymphocyte culture before testing. After lung transplantation, mean donor-specific IFN+ CD8 T-cell frequencies did not decrease as mean donor-specific LDA cytotoxic T lymphocyte precursor frequencies, whereas no difference was seen in pretransplantation samples or third-party-specific frequencies at both time points. Mean frequencies of IFN+ CD4 did not differ from helper T lymphocyte precursors at both time points, but frequencies did not correlate. CONCLUSIONS: The flow cytometry test allows a direct measurement of alloreactive T-cell frequencies and demonstrates a discrepancy between donor-specific IFN+ CD8 T-cell frequencies and LDA CLTp after transplantation. This may be a result of the existence of "functional diverse" alloreactive T cells or of activation-induced cell death of donor-reactive T cells during long (LDA) culturing, which is avoided in the flow cytometry test.

Donor brain death aggravates chronic rejection after lung transplantation in rats.

BACKGROUND: Many recipients of lung transplants from brain-dead donors develop bronchiolitis obliterans, a manifestation of chronic rejection. It has been shown that brain death increases inflammatory mediators and accelerates acute rejection in kidney, liver, and heart transplants. In this study, the authors investigated the hypothesis that brain death increases inflammatory mediators in the donor lung and subsequently aggravates chronic rejection of the lungs after transplantation in rats. METHODS: Brain death was induced in F344 rats by inflation of a subdurally placed balloon catheter. After 6 hr, donor lungs were assessed for influx of leukocytes, expression of cell adhesion molecules, and cytokine mRNA expression. For assessment of the lung after transplantation, lungs from brain-dead F344 rats were transplanted into WKY rats. Lung function after transplantation was monitored by chest radiographs during an observation period of 100 days. At the end of this period, the lungs were histologically examined; also, cytokine mRNA expression was measured. Lungs from ventilated living donors and living donors served as controls. RESULTS: After 6 hr of brain death, influx of polymorphonuclear cells and macrophages and expression of vascular cell adhesion molecule-1 in the donor lungs was increased. After transplantation at postoperative day 100, the lung function was significantly decreased compared with allografts from living donors. In the lung allografts from brain-dead donors, histologic symptoms of chronic rejection were obvious, including severe intimal hyperplasia but without bronchiolitis obliterans. Interleukin-2 mRNA was significantly increased in allografts from brain-dead donors compared with living donors. CONCLUSIONS: This study shows that brain death induces an inflammatory response in the donor lung and subsequently aggravates chronic rejection after transplantation. This may explain the clinical difference in long-term function between lungs from cadaveric donors and living donors.

Development of vasculature targeting strategies for the treatment of cancer and chronic inflammatory diseases.

Endothelial cells play a pathological role in cancer and chronic inflammation and are therefore attractive targets for therapeutic intervention. This review focuses on endothelial cell specific drug targeting strategies for the treatment of these diseases. The cellular and molecular processes involved in the activation of endothelial cells in angiogenesis and inflammation will be reviewed. Various target epitopes expressed by activated endothelium suitable for targeting purposes, design and development of drug-carrier complexes, drugs of interest which might interfere with endothelial cell activation, as well as in vitro and in vivo experimental approaches to study (intra) cellular drug delivery will be discussed.

Endothelial cells internalize and degrade RGD-modified proteins developed for tumor vasculature targeting.

Tumor vasculature can be targeted by peptides containing an RGD (Arg-Gly-Asp) sequence, which bind to alpha(v)beta3 and alpha(v)beta5 integrins on angiogenic endothelial cells. By covalently attaching cyclic RGD-peptides (cRGDfK) to a protein backbone, we prepared a multivalent peptide-protein conjugate with increased affinity for alpha(v)beta3/alpha(v)beta5 integrins. We demonstrated that RGDpep-protein conjugate bound to HUVEC, whereas the conjugate prepared with the control RAD peptide was devoid of any binding. RGDpep-protein conjugate was furthermore functional in inhibiting the adhesion of HUVEC to alpha(v)beta3/alpha(v)beta5 ligand vitronectin, and direct binding of the radiolabeled conjugate to HUVEC was inhibited by alpha(v)beta(3)/alpha(v)beta5-specific RGD peptides. Finally, RGDpep-protein conjugate was shown to be internalized and degraded by HUVEC, a process that could be inhibited by lysosomal degradation inhibitors chloroquine and ammonium chloride. This cellular handling was significantly influenced by the presence of cations, which strongly inhibited internalization. This is the first study that shows direct evidence that primary endothelial cells are capable of internalizing RGD-containing macromolecular proteins. This feature makes them attractive carriers for the intracellular delivery of potent anti-angiogenic drugs into endothelial cells for the treatment of cancer and chronic inflammatory diseases.

Cytokine production in natural killer cells and lymphocytes in pregnant women compared with women in the follicular phase of the ovarian cycle.

OBJECTIVE: To test whether peripheral natural killer (NK) cells, helper T cells, and cytotoxic lymphocytes of pregnant women shift from a type 1 cytokine production toward a type 2 cytokine production as compared with these cells in women in the follicular phase. DESIGN: Prospective study. SETTING: Outpatient clinic. PATIENT(S): Healthy nullipara at 30 weeks' amenorrhea and healthy nonpregnant women in their follicular phase. INTERVENTION(S): Samples of whole blood were stimulated with phorbol myristate acetate (PMA; Sigma Chemical Co., St. Louis, MO) and Ca-ionophore in the presence of monensin (Sigma). Lymphocytes were stained with alpha-CD3, alpha-CD8, and alpha-interferon gamma (IFN-gamma) alpha-interleukin 2 (IL-2), IL-4, or IL-10. Analysis was performed by flow cytometry. Statistical evaluation was done with the Mann-Whitney U test. MAIN OUTCOME MEASURE(S): Percentage NK cells, helper lymphocytes, and cytotoxic lymphocytes that were producing IFN-gamma, IL-2, IL-4, or IL-10. RESULT(S): There is a statistically significant decrease in the percentage of NK cells, and helper and cytotoxic lymphocytes that produced IFN-gamma in pregnant women when compared with women in the follicular phase. There is also a statistically significant decrease in the percentage of helper lymphocytes producing IL-2 in pregnant women compared with nonpregnant women. CONCLUSION(S): We found a decrease in type 1 cytokine production with no change in type 2 cytokine production after in vitro stimulation of "pregnant" NK cells and lymphocytes as compared with "nonpregnant" NK cells and lymphocytes. We suggest that NK cell and lymphocyte response are shifted away from a type 1 immune response during pregnancy.

Distribution patterns of the membrane glycoprotein CD44 during the foreign-body reaction to a degradable biomaterial in rats and mice.

Although biomaterials have been used in the clinical setting for a long time, little is known of the molecular mechanisms underlying the foreign-body reaction (FBR). A good understanding of these mechanisms is requisite for the controlled regulation of the FBR needed to prevent adverse tissue reactions and thus to improve the function of the biomaterial. Macrophages are essential in the inflammatory reaction in, as well as around, the implants, and they also are believed to initiate most of the adverse responses. Typically, during the FBR macrophages become activated and fuse into multinucleated giant cells (MnGCs). CD44, an integral membrane glycoprotein expressed on a broad spectrum of cell types, is involved in MnGC formation in vitro and in inflammation processes in general. In vivo it is not known whether CD44 is part of a specific protein machinery that enables macrophage fusion or whether it has additional functions in the FBR. In the present in vivo study, CD44 expression patterns were followed in rats and mice during the FBR to a degradable collagen type I biomaterial. We found that CD44 is upregulated on all migrating cells and on newly formed blood vessels at the onset of the FBR and that MnGCs, up to week 15 postimplantation, expressed CD44. Although no evidence was found that CD44 participates in macrophage fusion leading to multinucleation, it nevertheless may be an interesting target molecule for modulating the FBR in vivo, possibly by affecting cell activation, cell migration towards the biomaterial, vascularization, and MnGC formation.

Ongoing foreign body reaction to subcutaneous implanted (heparin) modified Dacron in rats.

Dacron-containing heart valve repair devices trigger chronic inflammation characterized by the presence of activated macrophages, foreign body giant cells, and capsule formation. Upon blood contact, proinflammatory proteins adsorb to the material and provide a substrate for monocyte binding and differentiation. Various heparin-coated polymers have been shown to reduce adsorption of proinflammatory proteins in vitro and in vivo. In this study, the effect of knitted, heparin-coated Dacron on the foreign body reaction was tested subcutaneously in rats. We hypothesized that the anti-inflammatory effect of heparin would reduce monocyte recruitment and differentiation and therefore limit the inflammatory reaction. An ongoing foreign body reaction, characterized by the presence of foreign body giant cells and high vascularization, was observed in uncoated as well as (heparin-)coated Dacron at up to 180 days of implantation. Also, a thin capsule was formed around each material up to this time. In conclusion, although heparin coatings might have an effect on the acute inflammatory response, we were not able to show a difference between heparin-coated and uncoated Dacron after 180 days' implantation in rats. Further research needs to be conducted to assess the difference in proinflammatory protein adsorption between the tested materials and the effect this has on the long-term foreign body reaction.

Serum insensitive, intranuclear protein delivery by the multipurpose cationic lipid SAINT-2.

Cationic liposomal compounds are widely used to introduce DNA and siRNA into viable cells, but none of these compounds are also capable of introducing proteins. Here we describe the use of a cationic amphiphilic lipid SAINT-2:DOPE for the efficient delivery of proteins into cells (profection). Labeling studies demonstrated equal delivery efficiency for protein as for DNA and siRNA. Moreover, proteins complexed with Saint-2:DOPE were successfully delivered, irrespective of the presence of serum, and the profection efficiency was not influenced by the size or the charge of the protein:cationic liposomal complex. Using beta-galactosidase as a reporter protein, enzymatic activity was detected in up to 98% of the adherent cells, up to 83% of the suspension cells and up to 70% of the primary cells after profection. A delivered antibody was detected in the cytoplasm for up to 7 days after profection. Delivery of the methyltransferase M.SssI resulted in DNA methylation, leading to a decrease in E-cadherin expression. The lipid-mediated multipurpose transport system reported here can introduce proteins into the cell with an equal delivery efficiency as for nucleotides. Delivery is irrespective of the presence of serum, and the protein can exert its function both in the cytoplasm and in the nucleus. Furthermore, DNA methylation by M.SssI delivery as a novel tool for gene silencing has potential applications in basic research and therapy.

Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis.

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.

Biodistribution studies of epithelial cell adhesion molecule (EpCAM)-directed monoclonal antibodies in the EpCAM-transgenic mouse tumor model.

The human pancarcinoma-associated epithelial cell adhesion molecule (EpCAM) (EGP-2, CO17-1A) is a well-known target for carcinoma-directed immunotherapy. Mouse-derived mAbs directed to EpCAM have been used to treat colon carcinoma patients showing well-tolerable toxic side effects but limited antitumor effects. Humanized or fully human anti-EpCAM mAbs may induce stronger antitumor activity, but proved to produce severe pancreatitis upon use in patients. To evaluate treatment-associated effects before a clinical trial, we have generated a transgenic mouse tumor model that expresses human EpCAM similar to carcinoma patients. In this study, we use this model to study the in vivo behavior of two humanized and one mouse-derived anti-EpCAM mAb, i.e., MOC31-hFc, UBS54, and MOC31. The pharmacokinetics and tissue distribution of the fully human mAb UBS54 and the mouse-derived MOC31 were largely the same after injection in tumor-bearing transgenic mice, whereas the molecularly engineered, humanized MOC31-hFc behaved differently. Injection of UBS54 and MOC31 resulted in significant, dose-dependent uptake of mAb in EpCAM-expressing normal and tumor tissues, accompanied by a drop in serum level, whereas injection of MOC31-hFc resulted in uptake in tumor tissue, limited uptake by normal tissues, and slow blood clearance. It is concluded that the EpCAM-transgenic mouse model provides valuable insights into the potential behavior of humanized anti-EpCAM mAbs in patients. mAbs sharing the same epitope and isotype but constructed differently were shown to behave differently in the model, indicating that the design of mAbs is important for eventual success in in vivo application.