Epithelial Glycoprotein-2 Research Articles

Derlin-1 and Stanniocalcin-1 are Differentially Regulated in Metastasizing Canine Mammary Adenocarcinomas

Derlin-1, stanniocalcin-1, epithelial glycoprotein-2 (EGP-2) and maspin are associated with the metastasis of human breast cancer cells. This study reports the potential role of these molecules in metastasis of canine mammary tumours. Laser microdissected tissue samples were prepared from normal canine mammary gland and from simple adenomas, adenocarcinomas and their lymph node metastases. The expression of genes encoding the molecules of interest in these tissues was determined by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Most adenomas displayed reduced expression of derlin-1 mRNA. Few adenocarcinomas overexpressed derlin-1 mRNA, but all lymph node metastases overexpressed this gene product. Stanniocalcin-1 mRNA was not expressed within adenomas and was reduced in adenocarcinomas and their lymph node metastases. EGP-2 gene expression did not differ between normal, benign and malignant neoplastic tissues. Maspin gene expression varied markedly among the tumours with reduced or increased expression compared with normal mammary gland. Taken together, these results suggest that malignant behaviour of canine mammary adenocarcinoma is associated with reduced transcription of the stanniocalcin-1 gene and overexpression of the derlin-1 gene.

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein‐2 promoter as a novel anti‐cancer treatment

Zinc finger protein transcription factors (ZFP-TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA-binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene-specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA-binding motifs. In this study, we engineered ZFP-TFs to downregulate the activity of the epithelial glycoprotein-2 (EGP-2) promoter. The protein EGP-2 is overexpressed in a wide variety of cancer types and EGP-2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP-2 expression by ZFP-TFs provides a novel anti-cancer therapeutic. Using a straightforward strategy, we engineered a 3-ZFP that could bind a 9 bp sequence within the EGP-2 promoter. After the addition of a repressor domain, this 3-ZFP-TF could efficiently downregulate EGP-2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP-2 promoter activity. To inhibit the endogenous EGP-2 promoter, we engineered 6-ZFP-TFs. Although none of the constructed ZFP-TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP-TFs are versatile bi-directional modulators of gene expression and downregulation of EGP-2 promoter activity using ZFP-TFs can ultimately result in a novel anti-cancer treatment.

Highly efficient and carcinoma-specific adenoviral replication restricted by the EGP-2 promoter

Although some successes have been reported using adenoviral vectors for the treatment of cancer, adenoviral cancer gene therapy is still hampered by the lack of sufficient tumor cell killing. To increase the efficiency, adenoviruses have been modified to replicate specifically in tumor tissues by using tumor specific promoters controlling genes essential for adenoviral replication. However, many conditionally replicating adenoviral vectors replicate in one tumor type only, which limits their application. The epithelial glycoprotein-2 (EGP-2) promoter is active in a broad variety of carcinomas, the most common type of cancer. We utilized this promoter to restrict adenoviral replication. In this report we demonstrate that the potency of the replication-competent adenovirus AdEGP-2-E1 to specifically lyse EGP-2 positive cells is comparable to wild-type adenovirus (AdWT). In addition, we show that in vivo AdEGP-2-E1 replicates as efficient as AdWT in EGP-2 positive tumor cells. On the contrary, in EGP-2 negative cell lines as well as in primary human liver samples, the replication was attenuated up to 4-log in comparison to wild-type virus. This report clearly shows the potency of the EGP-2 promoter to mediate highly efficient and specific adenoviral replication for carcinoma gene therapy.

1000. Down-Regulating the Epithelial Glycoprotein-2 Promoter Utilizing Engineered Zinc Finger Protein Transcription Factors

Top of pageAbstract Engineered zinc finger protein transcription factors (ZFP-TFs) are emerging as novel powerful tools to regulate endogenous gene expression. Zinc finger proteins consist of modular building blocks, with each domain recognizing 3-4 base pairs. Stitching these modular domains together results in the recognition of an extended DNA sequence, increasing the specificity of the target sequence. Specific amino acid positions within each domain recognize the DNA. Altering the amino acid residues at these positions therefore results in the recognition of different base pairs. A wide variety of effector domains can be attached to the DNA-binding zinc finger protein, for example to activate or inhibit gene transcription. In this study we utilized engineered ZFP-TFs to specifically inhibit the expression of the epithelial glycoprotein-2 (EGP-2). EGP-2 is expressed in the most prevalent types of carcinomas, including breast, ovarian, and colon cancer. Recently, others have demonstrated that down-regulation of EGP-2 using siRNA or ODNs resulted in a decrease in the migration, invasion, and proliferation potential of tumor cells. Therefore, inhibiting the expression of EGP-2 can provide a novel anti-cancer treatment for a broad range of tumor types. Down-regulating EGP-2 expression by targeting the mRNA requires the targeting of many molecules. Direct down-regulation of the EGP-2 expression at the DNA level using engineered ZFP-TFs can therefore be more effective. We engineered a zinc finger protein that specifically bound to a 9 base-pair target sequence in the EGP-2 promoter sequence. After attaching a gene-expression inhibition domain to the DNA-binding ZFP, co-transfection of EGP-2-positive cells with a plasmid expressing the engineered ZFP-TF and a plasmid containing the luciferase gene under the control of the EGP-2 promoter resulted in 60% inhibition of luciferase expression. To further increase the specificity for binding to the EGP-2 promoter, we engineered three different ZFP-TFs targeting 18 base pair sequences within the EGP-2 promoter. Co-transfection experiments demonstrated that the engineered ZFP-TFs caused up to 80% down-regulation of the EGP-2 promoter while no down-regulation of a non-target promoter was observed. Combining inhibition at the DNA level with inhibition at the RNA level using siRNA might even further enhance the down-regulation of EGP-2 promoter activity. In this study we have demonstrated that ZFP-TFs can be engineered to down-regulate the EGP-2 promoter, generating a novel therapeutic for the treatment of a broad variety of tumor types.

Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system

IntroductionIn view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Adenoviral vector mediated strategies for breast cancer gene therapy and virotherapy are a promising novel therapeutic platform for the treatment of breast cancer. However, the promiscuous tropism of adenoviruses (Ads) is a major concern. Employing tissue specific promoters (TSPs) to restrict transgene expression or viral replication is an effective way to increase specificity towards tumor tissues and to reduce adverse effects in non-target tissues such as the liver. In this regard, candidate breast cancer TSPs include promoters of the genes for the epithelial glycoprotein 2 (EGP-2), cyclooxygenase-2 (Cox-2), α-chemokine SDF-1 receptor (stromal-cell-derived factor, CXCR4), secretory leukoprotease inhibitor (SLPI) and survivin.MethodsWe employed E1-deleted Ads that express the reporter gene luciferase under the control of the promoters of interest. We evaluated this class of vectors in various established breast cancer cell lines, primary breast cancer cells and finally in the most stringent preclinical available substrate system, constituted by precision cut tissue slices of human breast cancer and liver.ResultsOverall, the CXCR4 promoter exhibited the highest luciferase activity in breast cancer cell lines, primary breast cancer cells and breast cancer tissue slices. Importantly, the CXCR4 promoter displayed a very low activity in human primary fibroblasts and human liver tissue slices. Interestingly, gene expression profiles correlated with the promoter activities both in breast cancer cell lines and primary breast cancer cells.ConclusionThese data suggest that the CXCR4 promoter has an ideal 'breast cancer-on/liver-off' profile, and could, therefore, be a powerful tool in Ad vector based gene therapy or virotherapy of the carcinoma of the breast.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.

In vitrocytotoxicity of carcinoma cells with 111In-labeled antibodies to HER-2

Antibodies conjugated to radionuclides emitting low-energy electrons, which include Auger electrons and some conversion electrons, were recently shown to efficiently kill cells bearing a high density of the antigen recognized. The primary purpose of this study was to determine if such killing could be obtained with anti-HER-2 antibodies conjugated to (111)In, using the chelator benzyl-diethylenetriaminepentaacetic acid, or (125)I. Target cells were the breast carcinoma SK-BR-3 and the ovarian carcinoma SK-OV-3.ip1. In preliminary experiments, antibody accumulation and catabolism during a 2- to 3-day incubation with antibody was investigated. The level of antibody uptake, in terms of molecules per cell, was high enough such that killing seemed feasible. With an (125)I label, but not an (111)In label, increasing the antibody concentration past a certain point caused a decrease in total antibody accumulation, which might be attributed to effects of antibody binding. To test for cytotoxicity, cells were incubated for 2 days with the labeled antibody, then assayed for colony-forming units with a limiting dilution assay. SK-BR-3 cells were strongly killed ( approximately 3 logs) by antibody 21.1, and 100% kill was obtained by combining two noncompeting antibodies to HER-2 (21.1 and 4D5). SK-OV-3.ip.1 cells were more resistant to killing, but use of the two-antibody mixture produced a surviving fraction of approximately 0.002. (111)In-labeled antibodies to other high-density antigens, epithelial glycoprotein-1 and epithelial glycoprotein-2, also killed these target cells. In contrast, unlabeled antibodies or a nonreactive-labeled antibody produced much less cytotoxicity. The same experiment with an (131)I label (a beta-particle emitter) resulted in much greater levels of nonspecific cytotoxicity and essentially no specific cytotoxicity. This approach may be effective for therapy of micrometastases.

314. The Epithelial Glycoprotein-2 Promoter for Highly Specific and Efficient Cancer Gene Therapy for a Broad Range of Tumors

Top of pageAbstract Adenoviral cancer gene therapy is hampered by the lack of specific and efficient transgene expression in tumor cells. Employing tissue specific promoters to restrict transgene expression will increase specificity towards the tumor tissue. In addition, specific promoters can be used to increase the efficiency of adenoviral gene therapy by controlling adenoviral replication. However, many specific promoters are restricted to specific tumor types. The tumor specific promoter epithelial glycoprotein-2 (EGP-2) is known to be active in various epithelial-derived cancers including ovarian, colon, and breast cancer and might therefore be a good candidate to target a broad range of tumor types. In this study, the EGP-2 promoter was employed to improve the therapeutic potential of adenoviral cancer gene therapy. The EGP-2 promoter demonstrated an outstanding specificity and activity profile in vivo. In mouse liver tissue, 3 log lower activity compared to the strong constitutively active CMV promoter was shown compared with only 1 log lower activity in EGP-2 positive tumor tissue. Subsequently, the EGP-2 promoter was employed to control the expression of the suicide gene thymidine kinase (AdEGP-2 SR39TK). In vitro selective cell killing after infection with AdEGP-2 SR39TK and GCV administration was seen in EGP-2 positive cell lines, but not in EGP-2 negative cells. After iv injection of AdEGP-2 SR39TK in mice an absence of liver toxicity was demonstrated after administration of GCV, in contrast to AdCMV SR39TK which showed clear liver damage. These promising findings are currently being explored in tumor bearing mice. To successfully treat cancer, all tumor cells have to be eradicated. However, even after local administration adenoviral vectors will infect only a limited amount of tumor cells. Therefore, to increase the efficiency of the adenovirus the EGP-2 promoter was utilized to control the E1A gene, which is essential for adenoviral replication. This conditionally replicating adenovirus (CRAd) should replicate selectively in EGP-2 positive tumor cells, causing cell lysis and spread into the tumor mass. This cycle of selective replication and cell lysis is continued until no further tumor cells can be infected. The EGP-2 based CRAd was as efficient in cell killing as wild-type adenovirus in EGP-2 positive cell lines. In contrast, in EGP-2 negative cell lines replication of the EGP-2 based CRAd was attenuated up to 2 log when compared with the wild-type adenovirus. Currently, the EGP-2 based CRAd is further investigated in tumor bearing mice. Overall, it can be concluded that the EGP-2 promoter demonstrates high specificity and efficiency in adenoviral context and can therefore be a powerful tool in cancer gene therapy for the treatment of a broad range of tumor types.

1073. Down-Regulation of Epithelial Glycoprotein-2 and VEGF-D by Engineered Zinc Finger Protein Transcription Factors

Engineered zinc finger protein (ZFP) transcription factors are emerging as powerful tools to regulate endogenous gene expression as they are capable of selectively down- or upregulate all (splice) variants of a gene (family), in contrast to siRNA. Zinc finger proteins consist of modular building blocks recognizing 3|[ndash]|4 base pairs each. Stitching these fingers together results in the recognition of an extended and therefore unique DNA sequence. The small size of these transcription factors allows easy manipulation for efficient delivery and multiple transcription factors can be delivered utilizing one vector. As the target DNA sequence does not have to be palindromic, it is feasible to specifically target virtually any DNA sequence in the human genome.

Evaluation of tumor-specific promoter activities in melanoma

Gene therapy is a novel therapy for melanoma. To date, however, there is still no powerful tumor specific promoter (TSP) to restrict the transgene expression in melanoma cells. In order to define a useful TSP for targeting in the context of melanoma gene therapy, four promoters, the cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (CXCR4), epithelial glycoprotein 2 (EGP-2), and survivin, were tested in both established melanoma cell lines and primary melanoma cells. We employed recombinant adenoviral vectors (reAds) each with a candidate TSP (the Cox-2, CXCR4, EGP-2, or survivin), a reporter luciferase gene, and a poly-A signal, all of which were inserted into the E1-deleted region. A reAdGL3Bcytomegalovirus (CMV), containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity. Luciferase activity was measured in multiple tumor cell lines and two primary melanoma cell cultures after infection with reAds. Human epithelial melanocytes, HEM, were used as normal control. In contrast to three other promoters, the survivin promoter exhibited the highest activities within both melanoma cell lines and primary melanoma cells, but not in HEMs. Additionally, the survivin promoter exhibited very low activities in major mouse organs including the liver, in vivo. EGP-2 is not active in melanoma; messenger RNA expressions were correlated to promoter activities both in melanoma cell lines and primary cell cultures. Thus, these data suggest that the survivin promoter achieved a 'tumor-on/liver-off' profile, and thus represents a potentially useful tumor-specific promoter with applications for transcriptional targeting of Ad vector-based cancer gene therapy or oncolysis to melanoma.

467. Cationic Liposome-Based Delivery of DNA Modulating Enzymes to Specifically Regulate Gene Expression in Carcinoma Derived Cell Lines

Gene therapy so far has not fulfilled its promise, mainly because efficiency and specificity of both the gene transferred and the transferring device are not sufficient enough. A new and potentially powerful way to treat diseases is to interfere with the endogenous gene expression of the gene of interest using DNA modifying enzymes, such as restriction enzymes and DNA-methyltransferases. These enzymes are commonly used in a wide range of genomic analysis, and by altering their specificity by coupling of gene-specific oligonucleotides or peptide-nucleic acids their specificity can be programmed at will. The delivery of these enzymes to the cell, however, remains a problem. Using the cationic liposomal device SAINT, we succeeded in the delivery of a wide range of proteins including the restriction enzyme PvuII and the methyltransferase M.SssI, which displayed functionality upon delivery, e.g. DNA fragmentation and silencing of transcription. We focused on the use of the methyltransferase enzyme M.SssI to silence the transcription of genes expressed by the human EGP-2 promoter. The epithelial glycoprotein 2 (EGP-2), also known as Ep-CAM or the pancarcinoma associated protein 17-1A, is expressed at high levels on the surface of most carcinomas. Despite the fact that EGP-2 is also expressed on normal simple epithelial tissue, EGP-2 is regarded as an attractive target for anti-cancer treatment strategies. We used this promoter to drive expression in an EGP-2 transgenic mouse model (Cancer Research, 2001) and to drive suicide genes to induce carcinoma specific cell death (submitted). Here we show that methylation is one of the mechanisms involved in the regulation of this carcinoma-specific promoter and that we can silence expression of genes driven by this promoter upon profection of the M.SssI enzyme. This is the first step in proofing that by targeting DNA manipulating enzymes expression of endogenous genes involved in diseases can be altered.

292. Modification of the p53 Transgene of a Replication-Competent Adenovirus Improves p53 Stability and Cancer Cell Killing

Adenoviruses are widely used as vectors to deliver genetic material into cells. Although these vectors are currently the most efficient vectors used, they are not specific and not efficient enough to result in a good clinical outcome in the treatment against cancer. To increase the specificity, a transgene can be placed under the control of a tumor specific promoter, such as the epithelial glycoprotein-2 (EGP2) promoter. This promoter is active in a broad range of epithelial derived cancers. The length of the EGP2 promoter fragment used in initial studies is 3.4 kb. This promoter fragment showed in adenoviral context high activity and selectivity when tested both in vitro and in vivo. However due to limited space in the adenoviral genome, this promoter fragment is too large for the incorporation of several expression cassettes.

293. The Epithelial Glycoprotein-2 Promoter Controls Selective and Efficient Killing of Epithelium Derived Cancer Cells

Top of pageAbstract Replication-competent adenoviruses have oncolytic properties, but this oncolytic effect is insufficient for successful cancer therapy. In order to improve the anti-tumor activity, a replication-competent adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle was previously constructed. p53 expression from this vector significantly improved tumor cell killing and viral spread (Hum Gen Ther 2002: 13, 859). However, the full extent of this effect may be limited by the expression of inhibitory adenoviral and cellular proteins. The goal of the current work is to further enhance the anti-tumor activity of the p53-expressing replicating vector. The adenoviral E1b-55kD protein and cellular mdm2 have been shown to bind and inactivate p53. E1b-55kD is an early viral protein and p53 expression from Adp53rc has been engineered to peak late in the viral life cycle. However, the effect of p53 may still be limited by an interaction with E1b-55kD and mdm2. We hypothesized that modification of the p53 transgene to prevent binding to E1b-55kD and mdm2 would further improve the oncolytic effect of the vector. Therefore, a new vector (Adp53 Δ23) that is identical to Adp53rc except for a change of amino acid 23 (from tryptophan to serine) within the E1b-55kD/mdm2 binding region of the p53 transgene was constructed. In contrast to wild-type p53, the modified p53 protein expressed from this vector did not co-precipitate with E1b-55kD. In addition, the half-life of the modified p53 protein was prolonged. After viral infection with Adp53rc and Adp53 Δ23 for 24 hours, new protein production was inhibited with cycloheximide and p53 levels were analyzed by immunoblotting. Wild-type p53 was rapidly degraded and almost undetectable at 1 hour. In contrast, the levels of the modified p53 did not substantially change for at least 24 hours. Expression of the modified p53 protein did not impair effective virus propagation. Replication of Adp53 Δ23, measured by a modified plaque assay, was similar to replication of Adp53rc and an identical p53-negative control virus (Ad-co). Survival of three cancer cell lines (H1299, A549, H116), evaluated by WST-1 assay, was substantially decreased after infection with Adp53 Δ23 compared to Adp53rc and Ad-co. In conclusion, modification of the p53 transgene of a replication-competent adenovirus improves p53 stability and cancer cell killing without impairment of viral replication.

An in vitro model for purging of tumour cells from ovarian tissue.

Cryopreservation and autografting of ovarian tissue may preserve fertility after cancer treatment, but could be hampered by tumour cell contamination. Epithelial tumour cell lysis can be obtained with cytotoxic T cell retargeting through the bispecific antibody BIS-1, with combined affinity for the T-cell receptor and epithelial glycoprotein-2 (EGP-2). Our aim was to study the concept of tumour cell purging in the setting of a suspension of ovarian tissue. Human ovarian tissue was brought into suspension by mechanical and enzymatic treatment. Cells of the MCF-7 breast cancer cell line and activated human lymphocytes were co- incubated for 4 h with or without BIS-1 and with or without ovarian suspension. After incubation, MCF-7 cell death and cell growth were evaluated by fluorescent cell detection and MTT assay, respectively. Ovarian tissue morphology was evaluated immunohistochemically. MCF-7 cell death and cell growth inhibition increased with increasing ratios of lymphocytes to MCF-7 cells. BIS-1 addition gave further augmentation, with a maximum depletion of growing MCF-7 cells of 89% (SD 11%) versus without BIS-1, 23% (SD 15%; P < 0.001). Follicles remained morphologically intact. Purging of added epithelial tumour cells from ovarian tissue with BIS-1 is possible in vitro. Morphologically, follicles remain intact after this procedure. This method may contribute to safer replacement of ovarian tissue in female cancer survivors.

Carcinoembryonic antigen mRNA analysis detects micrometastatic cells in blood from lung cancer patients.

The current authors previously identified circulating cells expressing carcinoembryonic antigen (CEA) messenger ribonucleic acid (mRNA) in 80% of lung cancer patients bearing distant metastases. The current study prospectively validated the data on a novel cohort and extended the study to other mRNAs expressed by neoplastic cells. CEA, cytokeratin 19 and 20, aldolase A and epithelial glycoprotein 2 (EPG2) mRNA was analysed by reverse transcriptase-polymerase chain reaction in circulating cells from 19 healthy controls, and in biopsies and blood at diagnosis from 32 lung cancer patients monitored for 24 months. Aldolase A and cytokeratin 19 mRNA occurred in circulating cells of all controls; cytokeratin 20 was not expressed by any lung cancer biopsy. EPG2 mRNA occurred in all biopsies but not in the patients' circulating cells. CEA mRNA occurred in 29/32 (90.6%) biopsies and in 17/32 mRNA samples from circulating cells from lung cancer patients. Of these positive patients 12/17 developed metastases within 9 months of mRNA analysis. Three positive patients died, one was lost to follow-up, and one did not develop metastases within 24 months. Of the negative patients 12/15 did not develop metastases during the 24-month follow-up; one patient was lost to follow-up, one did not express CEA, and another developed metastases. Unlike in other neoplasias, cytokeratin 19 and 20, aldolase A and epithelial glycoprotein 2 messenger ribonucleic acid are not useful for the detection of circulating cancer cells in lung cancer. Carcinoembryonic antigen messenger ribonucleic acid analysis in circulating cells helps to identify lung cancer patients at a greater risk of metastases.

Lineage-specific identification of nonhematopoietic neoplasms by flow cytometry.

To extend flow cytometry (FC) to the diagnosis of nonhematopoietic neoplasms, we have developed new flow cytometric assays to identify expression of cytokeratin, epithelial cell adhesion molecule (EpCAM)/epithelial glycoprotein-2, myogenin, and CD99. To validate these assays, we correlated the flow cytometric results with the histologic and immunohistochemical results on paraffin-embedded tissue in a series of 21 cases, including 17 carcinomas, 1 atypical carcinoid, 2 rhabdomyosarcomas, and 1 Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). Six of 7 assayed carcinomas and the carcinoid were positive for cytoplasmic cytokeratin by the flow cytometric assay. EpCAM was expressed by 11 of 12 carcinomas that were assayed by FC. Both rhabdomyosarcomas expressed myogenin by FC, and the ES/PNET case expressed CD99. Interestingly, the blast-associated antigen CD90 was expressed uniformly on the ES/PNET case and on subsets of cells in the rhabdomyosarcoma and carcinoma cases. Potential applications of the flow cytometric assay to nonhematopoietic neoplasms will include evaluating samples with limited material, monitoring disease persistence and recurrence in patients with previous diagnoses, and making rapid diagnoses in urgent cases.

Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells.

A nonviral gene delivery vector has been developed in our laboratory based on the cationic polymer, poly(2-(dimethylethylamino)ethyl methacrylate) (p(DMAEMA)). p(DMAEMA)-based polyplexes have been successfully used for the transfection of OVCAR-3 cells in vitro. However, these polyplexes were unable to transfect OVCAR-3 cells growing in the peritoneal cavity of nude mice after intraperitoneal administration, which could be ascribed to inactivation by components (including hyaluronic acid) present in the tumor ascitic fluid. The present work aimed at (a) protecting p(DMAEMA)-based polyplexes against destabilization or inactivation by polyanions such as hyaluronic acid present in tumor ascitic fluid and (b) enhancing cellular uptake of the protected p(DMAEMA)-based polyplexes by targeting with antibody Fab' fragments. To fulfill these requirements, we have developed a detergent removal method to coat polyplexes with anionic lipids. With this method, spherical particles of approximately 125 nm, which were protected from destabilization by polyanions, were obtained. More importantly, the transfection efficiency of lipopolyplexes was unaffected in the presence of hyaluronic acid, indicating that lipid coating of polyplexes protects against destabilization by hyaluronic acid. By conjugating antibody Fab' fragments directed against the epithelial glycoprotein-2 to the lipidic surface of these lipopolyplexes, target cell-specific transfection of OVCAR-3 cells could be obtained in vitro.

Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of “guided selection” to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the V H of MOC-31 was combined in Fab format with a human V LC L library, a small panel of human light chains was identified, originating from a segment of the VκIII family, whereas the MOC-31 V L is more homologous to the VκII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V L of C3 with a human V H library, while retaining the V H CDR3 of MOC-31, clones were selected using human V H genes originating from the rarely used V H7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the “MOC-31“-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both V L and V H of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V H CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting.

Specific targeting of EGP-2+ tumor cells by primary lymphocytes modified with chimeric T cell receptors.

A promising strategy for cancer treatment is adoptive immunotherapy with gene-modified lymphocytes expressing a chimeric T cell receptor (cTCR) that directs tumor targeting and stimulates T cell effector functions. In this study, the activities of two novel cTCR molecules (GAgamma and GAHgamma) were investigated. Both encode a single-chain variable fragment (scFv) derived from the monoclonal antibody (MAb) GA733.2, which binds the epithelial glycoprotein 2 (EGP-2) overexpressed on a variety of human carcinomas. In the GAgamma cTCR, the scFv is directly fused to the transmembrane/cytoplasmic portions of the immunoglobulin Fc receptor (Ig FcRI) gamma subunit, which mediates T cell signaling. GAHgamma possesses an extracellular spacer composed of the CD8alpha immunoglobulin hingelike domain inserted between the scFv and gamma chain. Activated T cells (ATCs), stimulated ex vivo using anti-CD3 MAb, were derived from either healthy donors or patients and transduced with recombinant retrovirus encoding the respective GA cTCR molecules. After culture expansion for 14 days, GAgamma-modified ATCs demonstrated enhanced targeting and lysis of EGP-2+ colon cancer cells and increased cytokine secretion. Cells transduced with the GAHgamma cTCR displayed specific lytic activity that was about twofold greater than that of GAgamma-ATCs and produced significantly more cytokine. In addition, reactivation of GAHgamma-ATC with anti-CD3 MAb prior to addition to EGP-2+ tumor target induced a further increase in lytic activity. Because the activation status influences T cell antitumor functions, our data suggest that reactivation prior to adoptive transfer would improve the clinical efficacy of GAHgamma-modified ATCs.

Photochemical internalisation increases the cytotoxic effect of the immunotoxin MOC31-gelonin

Photochemical internalisation (PCI) was recently demonstrated as a unique procedure for site-specific delivery of several types of membrane impermeable macromolecules from endocytotic vesicles to the cytosol (Berg et al., 1999). The technology is based on the cytosolic release of endocytosed macromolecules from endosomes and lysosomes upon exposure of cells to photosensitising compounds, which became localised to these vesicles, and light. In our study the possibility to increase the cytotoxic effect of the immunotoxin MOC31-gelonin by PCI was examined. The type I ribosome-inactivating protein gelonin was covalently linked to the monoclonal IgG1 antibody MOC31, directed against epithelial glycoprotein-2 (EGP-2), an antigen expressed on most carcinoma cells. Five different cell lines, of which 4 expressed EGP-2, were treated with MOC31-gelonin and endosomal and lysosomal localising photosensitisers, followed by exposure to light. Insignificant cytotoxicity of the MOC31-gelonin was observed when the cells were incubated with the immunotoxin alone. However, in combination with endosomal and lysosomal localising photosensitizers, we demonstrate synergistic toxic effect of the MOC31-gelonin conjugate in a light-dependent manner. Our results indicate that PCI is a promising tool for increasing the cytotoxicity of immunotoxins, which is important for further improvement of the PCI concept towards possible use in cancer therapy.