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.

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.

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.

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.

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.

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.

Kinetics of US28 gene expression during active human cytomegalovirus infection in lung-transplant recipients.

Targeting viral proteins early during infection may limit exacerbation of human cytomegalovirus infection. The viral chemokine-receptor homologue US28 interferes with leukocyte trafficking and, possibly, viral replication. Because US28 molecules are abundant on the surface of infected cells, this homologue is a potential target for antiviral therapy. To assess the relationship between US28 and disease activity, we measured, by quantitative reverse-transcription polymerase chain reaction, the levels of US28 and immediate-early (IE) 1 gene transcripts in the blood of lung-transplant recipients. We found that, during primary and secondary infection, the IE1 and US28 genes have early transcription kinetics and are expressed at similar levels. This may render US28 an attractive target for antiviral therapy.

Use of sequence variation in three highly variable regions of the mitochondrial DNA for the discrimination of allogeneic platelets.

BACKGROUND: Human mitochondrial DNA (mtDNA) polymorphisms can be used to detect allogeneic transfused platelets. To increase the number of informative polymorphisms we investigated three hypervariable regions (HVR1, HVR2, and HVR3) within the displacement loop (D-loop) region of the mtDNA. STUDY DESIGN AND METHODS: mtDNA was obtained from 119 unrelated blood donors. Forward and reverse primers were designed and conditions optimized to amplify and sequence the template mtDNA by dye terminator cycle sequencing. RESULTS: We established a sequencing protocol for all three HVRs of the mtDNA. Polymorphic sites were found in all three regions: 66 in HVR1, 44 in HVR2, and 18 in HVR3. Combining the sequence information of HVR1, -2, and -3 resulted in 105 different genotypes of which 95 were unique. We were able to discriminate between two randomly chosen individuals with a random match probability of 1.2 percent. CONCLUSION: The D-loop region of mtDNA contains a wealth of informative molecular markers for chimerism and survival studies after transfusions of cellular blood components.

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.

CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia.

Agonistic anti-Fas antibodies and multimeric recombinant Fas ligand (FasL) preparations show high tumoricidal activity against leukemic cells, but are unsuitable for clinical application due to unacceptable systemic toxicity. Consequently, new antileukemia strategies based on Fas activation have to meet the criterion of strictly localized action at the tumor-cell surface. Recent insight into the FasL/Fas system has revealed that soluble homotrimeric FasL (sFasL) is in fact nontoxic to normal cells, but also lacks tumoricidal activity. We report on a novel fusion protein, designated scFvCD7:sFasL, that is designed to have leukemia-restricted activity. ScFvCD7:sFasL consists of sFasL genetically linked to a high-affinity single-chain fragment of variable regions (scFv) antibody fragment specific for the T-cell leukemia-associated antigen CD7. Soluble homotrimeric scFvCD7:sFasL is inactive and acquires tumoricidal activity only after specific binding to tumor cell-surface-expressed CD7. Treatment of T-cell acute lymphoblastic leukemia (T-ALL) cell lines and patient-derived T-ALL, peripheral T-cell lymphoma (PTCL), and CD7-positive acute myeloid leukemia (AML) cells with homotrimeric scFvCD7:sFasL revealed potent CD7-restricted induction of apoptosis that was augmented by conventional drugs, farnesyl transferase inhibitor L-744832, and the proteasome inhibitor bortezomib (Velcade; Millenium, Cambridge, MA). Importantly, identical treatment did not affect normal human peripheral-blood lymphocytes (PBLs) and endothelial cells, with only moderate apoptosis in interleukin-2 (IL-2)/CD3-activated T cells. CD7-restricted activation of Fas in T-cell leukemic cells by scFvCD7:sFasL revitalizes interest in the applicability of Fas signaling in leukemia therapy.

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 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.

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.

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR.

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

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.

The role of B cell-mediated T cell costimulation in the efficacy of the T cell retargeting bispecific antibody BIS20x3.

In this study, we investigated the role of the naturally occurring B cell-mediated T cell costimulation in the antitumor efficacy of the bispecific Ab BIS20x3. BIS20x3 has a dual specificity for both CD20 and CD3 and has previously been shown to effectively direct the lytic potential of cytolytic T cells toward malignant, CD20(+) B cells. BIS20x3 instigated T cell-B cell interaction caused a dose-dependent activation of T cells that was 30 times stronger when compared with T cell activation induced by monovalent anti-CD3 Abs. The activation of T cells by BIS20x3 and B cells appeared functional and resulted in the rapid induction of high lytic potential in freshly isolated peripheral T cells. BIS20x3-mediated T cell-B cell interaction resulted in a significant up-regulation of ICAM-1 on B cells and the activation of T cells was found to be dependent on the interaction of ICAM-1 with LFA-1 and trans-activation by the NF-kappaB pathway. Also, the lytic potential of freshly isolated T cells activated via BIS20x3 appeared to be dependent on NF-kappaB signaling in the target B cells. Interestingly, the costimulatory signaling effects described in this study appeared specifically related to the targeting against CD20 because targeting against CD19, by a CD3xCD19-directed bispecific Ab, was significantly less effective in inducing T cell activation and T cell-mediated B cell lysis. Together these results demonstrate that the malignant B cells actively contribute to their own demise upon CD20-directed bispecific Ab-mediated T cell targeting.

RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes toward alphavbeta3-expressing endothelial cells.

To redirect the lytic activity of cytotoxic T lymphocytes (CTL) toward tumor vascular endothelial cells, we prepared bifunctional proteins with specificity for both alphavbeta3 and CD3. Monocyclic RGD peptides (cRGDfK) were covalently coupled to an anti-CD3 monoclonal antibody at different peptide:protein ratios. The resulting RGDpep-anti-CD3 conjugates bound specifically to alphavbeta3-expressing endothelial cells. Displacement studies with radiolabeled alphavbeta3 ligand demonstrated that the alphavbeta3 binding affinity of RGDpep-anti-CD3 conjugates was elevated as compared to the non-conjugated RGDpep. IC50 values ranged from 150-1.1 nM, depending on the amount of coupled RGDpep molecules per antibody molecule. RGD modification did not affect the ability of anti-CD3 antibodies to bind to CTL. Furthermore, RGDpep-anti-CD3 was fully capable of activating T cells upon CD3 binding as was shown in a Jurkat/NFAT reporter-gene activation assay. All RGDpep-anti-CD3 conjugates were able to induce RGDpep, CD3-dependent lysis of human primary endothelial cells by anti-CD3/IL-2 activated human peripheral blood mononuclear cells (PBMC), with a significant induction of cytotoxicity observed at an E/T ratio as low as 10. Redirecting cytolytic activity reached up to 50% cytotoxicity using the conjugate with the highest RGD peptide load. Combining the good accessibility of tumor blood vessel endothelium for CTL with the efficiency of target cell killing warrants further investigations on anti-tumor effects of this type of conjugates in vivo.

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.

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.