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David T Curiel

Publications and source records attributed to David T Curiel.

At least 163 records · Page 9Linked to original sources
PubMed2002

Construction and characterization of an intracellular single-chain human antibody to hepatitis C virus non-structural 3 protein.

BACKGROUND/AIMS: We developed a single-chain antibody fragment (scFv) to the non-structural 3 protein (NS3) of hepatitis C virus (HCV) and tested its ability to interfere with the HCV replication cycle in infected hepatocytes. METHODS: The variable regions of the human monoclonal antibody CM3.B6 that recognizes a conformational epitope within the helicase domain of NS3 were introduced into adenoviral vectors for expression in mammalian hepatocytes. Expression and binding properties of the scFv were analyzed by immunological assays. Effects of intracellular expression of the scFv on HCV replication were assessed in primary hepatocytes isolated from explanted livers of patients with chronic HCV infection by reverse transcription-polymerase chain reaction. RESULTS: Transduction of HepG2 cells by the recombinant adenoviruses resulted in stable, efficient expression of scFv in the cytoplasm that was non-toxic to the cells. The scFv specifically bound to its cognate antigen. Significantly, intracellular expression of scFv resulted in a decrease in HCV genomic RNA in HCV infected hepatocytes. CONCLUSIONS: These results indicate that specific binding of a scFv to NS3 may inhibit one or more functions of this essential viral protein thus interfering with the HCV replication cycle.

Adenoviridae↗
PubMed2002

Genetically modified adenovirus vector containing an RGD peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long-term euglycemia (>250d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin-producing cells and adequate long-term function of the p-cell after transplantation.

Adenoviridae↗
PubMed2002

New generation adenoviral vectors improve gene transfer by coxsackie and adenoviral receptor-independent cell entry.

Adenoviral (Ad) vectors possess many attributes that have lead to their use as gene delivery agents in human clinical trials. However, gene transfer efficiency has generally been less than that needed for meaningful clinical responses. The restricted tropism of the virus for its native receptor, the coxsackie and adenoviral receptor (CAR), is emerging as a key limitation to the use of these agents. By developing strategies to achieve Ad infection via alternate receptor pathways, enhanced and more specific gene delivery can be achieved. This new generation of tropism-modified agents holds promise for the improved clinical utility of Ad vectors for gene therapy.

Adenoviridae↗
PubMed2002

Cytoprotection of pancreatic islets before and early after transplantation using gene therapy.

Pancreatic islet transplantation (PIT) is an attractive alternative to insulin-dependent diabetes treatment but is not yet a clinical reality. The first few days after PIT are characterized by substantial pancreatic islet dysfunction and death. Apoptosis has been documented in PI after extracellular matrix removal, during culture time, after exposure to proinflammatory cytokines, hypoxic conditions before islet revascularization, and rejection. Targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, over-expression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation into diabetic SCID mice. Genetic modification of PI also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass post-transplant and reduce the high islet requirement currently needed for successful stable reversal of insulin-dependent diabetes [1, 2].

Animals↗
PubMed2002

Midkine promoter-based adenoviral suicide gene therapy to midkine-positive pediatric tumor.

BACKGROUND/PURPOSE: Suicide gene therapy based on the delivery of the herpes simplex virus thymidine kinase gene combined with ganciclovir (HSV-tk/GCV) is a promising approach for cancer treatment. Adenoviral (Ad) vectors are useful gene delivery vehicles for this approach; however, because these agents possess a high natural tropism for the liver, systems must be designed to avoid potential hepatotoxicity induced by expression of the therapeutic gene in this organ. It has been reported that Wilms' tumors or neuroblastomas express a high level of midkine (MK), a heparin-binding growth factor. In addition, no MK expression is observed in mouse or human liver. The authors investigated the application of MK promoter-based adenoviral gene therapy for MK-positive tumors, especially Wilms' tumors or neuroblastomas, and have shown that the MK promoter retains its fidelity in the adenoviral context, having low activity in liver and high activity in MK-positive tumor cells. We present herein the efficacy of in vivo tumor regression as well as prevention of lethal hepatic toxicity by using the MK promoter in an Ad vector-based HSV-tk/GCV treatment approach. METHODS: Ad vectors, AdMKTK or AdCMVTK, encoding HSV-tk under the control of the MK or cytomegalovirus promoters, respectively, were injected systemically into mice, then hepatotoxicity and survival was monitored after GCV administration. In concurrent studies, the therapeutic impact of AdMKTK/GCV versus AdCMVTK/GCV on subcutaneous G-401 Wilms' tumors in nude mice was assessed. RESULTS: By day 8 of systemic viral treatment, 4 of 5 mice treated with AdCMVTK/GCV had died, whereas all mice treated with AdMKTK/GCV survived at least 10 days. In the subcutaneous tumor study, equivalent regression of tumor was seen in the group that received AdMKTK as the group that received AdCMVTK intratumoral injection. CONCLUSION: These data indicate that the MK promoter-based adenoviral suicide gene therapy is a unique therapeutic candidate for MK-positive tumors, Wilms' tumors, or neuroblastomas, by virtue of its mild hepatotoxicity and high efficacy against MK-positive tumors.

Adenoviridae↗
PubMed2002

Hepatic DR5 induces apoptosis and limits adenovirus gene therapy product expression in the liver.

A major limitation of adenovirus (Ad) gene therapy product expression in the liver is subsequent elimination of the hepatocytes expressing the gene therapy product. This elimination is caused by both necrosis and apoptosis related to the innate and cell-mediated immune response to the Ad. Apoptosis of hepatocytes can be induced by the innate immune response by signaling through death domain receptors on hepatocytes including the tumor necrosis factor alpha (TNF-alpha) receptor (TNFR), Fas, and death domain receptors DR4 and DR5. We have previously shown that blocking signaling through TNFR enhances and prolongs gene therapy product expression in the liver. In the present study, we constructed an Ad that produces a soluble DR5-Fc (AdsDR5), which is capable of neutralizing TNF-related apoptosis-inducing ligand (TRAIL). AdsDR5 prevents TRAIL-mediated apoptosis of CD3-activated T cells and decreases hepatocyte apoptosis after AdCMVLacZ administration and enhances the level and duration of lacZ transgene expression in the liver. In addition to blocking TRAIL and directly inhibiting apoptosis, AdsDR5 decreases production of gamma interferon (IFN-gamma) and TNF-alpha and decreases NK cell activation, all of which limit Ad-mediated transgene expression in the liver. These results indicate that (i) AdsDR5 produces a DR5-Fc capable of neutralizing TRAIL, (ii) AdsDR5 can reduce activation of NK cells and reduce induction of IFN-gamma and TNF-alpha after Ad administration, and (iii) administration of AdsDR5 can enhance Ad gene therapy in the liver.

Adenoviruses, Human↗
PubMed2002

Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX.

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.

Adenoviruses, Human↗
PubMed2002

Modulation of adenovirus vector tropism via incorporation of polypeptide ligands into the fiber protein.

The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virion's integrity.

Adenoviruses, Human↗
PubMed2002

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

Base Sequence↗
PubMed2002

Construction and characterization of adenovirus serotype 5 packaged by serotype 3 hexon.

Adenovirus serotype 5 (Ad5) has great potential for gene therapy applications. A major limitation, however, is the host immune response against Ad5 infection that often prevents the readministration of Ad5 vectors. In this regard, the most abundant capsid protein, hexon, has been implicated as the major target for neutralizing antibodies. In this study, we sought to escape the host neutralization response against Ad5 via hexon replacement. We constructed a chimeric adenovirus vector, Ad5/H3, by replacing the Ad5 hexon gene with the hexon gene of Ad3. The chimeric viruses were successfully rescued in 293 cells. Compared to that for the control Ad5/H5, the growth rate of Ad5/H3 was significantly slower and the final yield was about 1 log order less. These data indicate that the Ad3 hexon can encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficacy of Ad5/H3 in HeLa cells was also lower than that of Ad5/H5. Furthermore, we tested the host neutralization responses against the two viruses by using C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the immunized mice did not cross-neutralize each other in the context of in vitro infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of viruses also did not prevent subsequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5. This strategy may therefore be used to achieve the repeated administration of Ad5 in gene therapy applications.

Adenoviridae↗
PubMed2002

Artificial extension of the adenovirus fiber shaft inhibits infectivity in coxsackievirus and adenovirus receptor-positive cell lines.

Recent studies demonstrate that virus-cellular receptor interactions are not the sole determinants of adenovirus (Ad) tropism. It has been shown that the fiber shaft length, which ranges from 6 to 23 beta-repeats in human Ads, also influences viral tropism. However, there is no report that investigates whether artificial extension of the shaft alters the infectivity profile of Ad. Therefore, we constructed Ad serotype 5 (Ad5) capsid-based longer-shafted Ad vectors by incorporating Ad2 shaft fragments of different lengths into the Ad5 shaft. We show that "longer-shafted" Ad vectors (up to 32 beta-repeats) could be rescued. We also show that longer-shafted Ad vectors had no impact on knob-CAR (coxsackievirus and Ad receptor) interaction compared to wild-type Ad. Nevertheless, gene transfer efficiencies of longer-shafted Ad vectors were lower in CAR-positive cell lines compared to wild-type Ad. We suggest that artificial extension of the shaft can inhibit infectivity in the context of CAR-positive cell lines without modification of knob-CAR interaction.

Adenoviruses, Human↗
PubMed2002

Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice.

PURPOSE: To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. MATERIALS AND METHODS: A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. RESULTS: Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose. CONCLUSION: The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector.

Adenoviridae↗
PubMed2002

Converting nonhuman primate dendritic cells into potent antigen-specific cellular immunosuppressants by genetic modification.

T cell depletion plus donor bone marrow cell (BMC) infusion induces long-term kidney allograft survival in a limited number of rhesus macaque recipients. Therefore, there is a need to enhance the tolerogenic activity of donor BMCs. The tolerogenic effect of donor BMCs is ascribed to a veto activity, mediated by a CD8+ subset that upregulates immunoregulatory effector molecules, transforming growth factor-beta1 (TGF-beta), and FasL, after interaction with donor-reactive cytotoxic T lymphocyte precursors (CTLp), leading to clonal inactivation/deletion of donor-reactive CTLp. Of note, the receptors for TGF-beta1- and FasL-induced signal transduction are upregulated in activated T cells. Since mature dendritic cells (DCs) are exceptionally efficient activators of T cells, we postulated that mature DCs modified to overexpress TGF-beta1 and FasL might exert potent veto (i.e., inactivating/deleting) activity independent of CD8 expression. A fusion protein comprising antihuman CD40 single-chain antibody and soluble coxsackie-adenovirus receptor enabled high-efficiency transduction of rhesus monocyte-derived DCs (Rh MDDCs) by recombinant adenovirus (Ad). Mature Rh MDDCs transduced with Ad encoding active TGF-beta1 retained a mature phenotype yet exhibited potent alloantigen-specific cellular immunosuppression. Such modified MDDCs have the potential to promote tolerance induction to allografts in vivo.

Animals↗
PubMed2002

Genetically engineered intracellular single-chain antibodies in gene therapy.

The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins.

Antibodies↗
PubMed2002

Gene therapy for meningioma: improved gene delivery with targeted adenoviruses.

OBJECT: Due to their surgical inaccessibility or aggressive behavior, some meningiomas cannot be cured with current treatment strategies. Gene therapy is an emerging strategy for the treatment of brain tumors, which the authors investigated to determine whether adenoviruses could be used for gene transfer in meningioma cells. METHODS: The presence of the high-affinity Coxsackievirus and adenovirus receptor (CAR) for adenovirus type 5, as well as endothelial growth factor receptor (EGFR) and alpha, integrins (ITGAVs), were analyzed in primary tumors by using immunohistochemical studies and in primary meningioma cell cultures by using fluorescence-activated cell sorting. Targeting of adenoviruses to EGFR was achieved using bispecific antibodies, whereas targeting of adenoviruses to the ITGAVs was accomplished by insertion of an RGD (arginine-glycine-aspartic acid) motif in the adenovirus fiber H1 loop. Gene transfer efficiency of untargeted and targeted vectors was compared in primary cell cultures and in spheroids derived from patients' resected tumor material. The presence of CARs was observed in all tumors and in all but one of the derived primary meningioma cells. The higher expression of EGFRs and ITGAVs indicated that these receptors could be used as alternative targets to redirect the adenoviruses. Redirection of adenoviruses to the EGFRs or integrins enhanced gene transfer threefold (range two-sevenfold) for EGFRs in primary meningioma cells and ninefold (range three-23-fold) for integrins (p = 0.002, analysis of variance). The effect of adenovirus targeting was confirmed in spheroids composed of primary meningioma cells. CONCLUSIONS: Gene transfer with adenoviruses targeted to tumor-specific receptors is very effective in primary meningioma cells and spheroids. These vectors are promising agents for gene therapy of meningiomas.

Adenoviridae↗
PubMed2002

Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells.

Gene delivery efficiency in clinical cancer gene therapy trials with recombinant adenoviruses (Ads) based on serotype 5 (Ad5) has been limited partly because of variable expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human primary cancer cells. As a means of circumventing CAR deficiency, Ad vectors have been retargeted by creating chimeric fibers possessing knob domains of alternate Ad serotypes. In this study, we have constructed an Ad5-based vector, Ad5/3luc1, with a chimeric fiber protein featuring a knob domain derived from Ad3. This virus is retargeted to the Ad3 receptor and, therefore, has different tissue tropism. A novel knob binding assay was used to measure expression of CAR and the Ad3 receptor. Further, to evaluate the correlation of receptor expression and infectivity by Ad, a panel of ovarian cancer cell lines and purified primary cancer cells were infected with Ad5luc1 and Ad5/3luc1 at 50, 200, and 1000 viral particles/cell. Our results confirm that Ad5/3luc1 is retargeted to the Ad3 receptor. Furthermore, the Ad3 receptor is present at higher levels than CAR on ovarian adenocarcinoma cells. Also, the amount of binding to primary receptor appears to be the major factor determining the efficiency of transgene expression. The Ad5/3 chimera displays enhanced infectivity for ovarian cancer cell lines and purified primary tumor cells, which could translate into increased efficacy in clinical trials.

Adenocarcinoma↗
PubMed2002

Safety and immunogenicity of a DNA vaccine encoding carcinoembryonic antigen and hepatitis B surface antigen in colorectal carcinoma patients.

Despite an abundance of preclinical data, relatively little is known regarding the efficacy of DNA vaccination in humans. Here, we present results from a dose-escalation clinical trial of a dual expression plasmid encoding carcinoembryonic antigen (CEA) and hepatitis B surface antigen (HBsAg) in 17 patients with metastatic colorectal carcinoma. CEA was selected as a prototypic tumor-associated self-antigen, and the HBsAg cDNA was included as a positive control for immune response to the DNA vaccine without relying upon breaking tolerance to a self-antigen. Groups of 3 patients received escalating single i.m. doses of the DNA vaccine at 0.1, 0.3, and 1.0 mg. Subsequent groups of 3 patients received three repetitive 0.3- or 1.0-mg doses at 3-week intervals. A final group of 2 patients received three repetitive 2.0 mg doses at 3-week intervals. Toxicity was limited to transient grade 1 injection site tenderness, fatigue, and creatine kinase elevations, each affecting a minority of patients in a non-dose-related manner. Repetitive dosing of the DNA vaccine induced HBsAg antibodies in 6 of 8 patients, with protective antibody levels achieved in four of these patients. CEA-specific antibody responses were not observed, but 4 of 17 patients developed lymphoproliferative responses to CEA after vaccination. No objective clinical responses to the DNA vaccine were observed among this population of patients with widely metastatic colorectal carcinoma. Nevertheless, this pilot trial has provided encouraging human immune response data in support of this vaccine technology.

Adenocarcinoma↗