The immunogenic properties of plasmid DNA and recombinant adenovirus (Ad) encoding the carcinoembryonic antigen (CEA) were examined in mice by measuring both the amplitude and type of immune response, and the immunogenicity of codon usage optimized cDNA encoding CEA (CEAopt) was assessed both in C57Bl/6 and CEA transgenic mice. Vectors were injected into quadriceps muscle either alone or in combination, and plasmid DNA was electroporated to enhance gene expression efficiency and immunogenicity. Injection of plasmid pVIJ/CEA followed by Ad-CEA boost elicited the highest amplitude of both CD4 1 and CD8 1 T-cell response to the target antigen, measured by both IFNc-ELIspot assay and intracellular staining. Vectors carrying cDNA of CEAopt expressed a greater amount of the CEA protein than their wild-type counterparts, and this enhanced expression was associated with greater immunogenicity. Both CD4 1 and CD8 1 T-cell epitopes were mapped in the C-terminal portion of the protein. In CEA transgenic mice, only immunization based on repeated injections of pVIJ/ CEAopt followed by Ad-CEAopt was able to elicit a CEA-specific CD8 1 T-cell response, whereas the wild-type vectors did not break tolerance to this target antigen. MC38-CEA tumor cells injected s.c. in CEA transgenic mice vaccinated with CEAopt vectors exhibited delayed growth kinetics. These studies demonstrate that this type of genetic vaccine is highly immunogenic and can break tolerance to CEA tumor antigen in CEA transgenic mice. ' 2005 Wiley-Liss, Inc.Key words: CEA; DNA electroporation; adenovirus Despite improvements in prevention, early detection and treatment, the possibility of curing many cancer patients remains elusive. Thus, cancer continues to be a largely unmet medical need for which more efficient therapeutic strategies must be developed. 1 Particular attention has been given to active specific immunotherapy of cancer, whereby patients are immunized against antigens presented by tumor cells. In fact, experimental and clinical evidence have demonstrated the critical role played by the cellular and humoral responses in controlling tumor growth and metastasis. 2 Many of these therapies are specifically targeted to tumorassociated antigens among which carcinoembryonic antigen (CEA) is a frequent example due to its ectopic and deregulated expression in a large percentage of adenocarcinomas. 3 Human CEA is the prototypic member of the human CEA family, a group of highly glycosylated homotypic/heterotypic cell surface intracellular adhesion molecules, and part of the immunoglobulin gene superfamily. CEA is widely used as human tumor marker, is expressed mostly in the gastrointestinal tract and is overexpressed in many human cancers, including epithelial tumors originating from the gastrointestinal tract, lung, thyroid, breast, prostate, cervix and ovaries. 4 CEA has been the focus of extensive preclinical and clinical investigation aimed at developing a CEA-specific vaccine with a therapeutic impact on tumor progression. 5 In this context, genetic vacc...
Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.
The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.
IntroductionAttempts to treat experimental tumors with adoptive T lymphocyte transfer started in 1960, soon after the demonstration that the cellular arm of the immune system was responsible for tissue rejection. 1 The translation of this approach to the clinic, however, became possible when human tumor-infiltrating lymphocytes (TILs) could be expanded in vitro and injected back into cancer patients. 2 Only recently, however, an objective cancer regression was achieved in approximately 50% of patients with metastatic tumor after the introduction of host preconditioning by lymphodepletion before the treatment, followed by high-dose interleukin-2 (IL-2) to sustain in vivo expansion of T cells. 3,4 So far, this approach has been limited to melanoma patients, from whom TILs of known specificity are easily cultured in vitro, whereas for tumors other than melanoma the rescue of TILs is problematic.Telomerase is considered an attractive target as universal antigen for the immunotherapy of cancer. Telomerase reverse transcriptase (TERT) is the protein component of the telomerase complex responsible for elongation of telomeres and plays an essential role in sustaining cancerous cell proliferation. 5 TERT is silent in most human somatic tissues but reactivated in 85% of tumors. Active immunization against telomerase and cancer vaccination clinical trials have been attempted that demonstrate little toxicity, but results concerning therapeutic efficacy are not conclusive. 6,7 Although T lymphocytes against telomerase were isolated and extensively characterized in vitro, 8,9 the use of adoptive cell therapy (ACT) with telomerase-specific lymphocytes has never been investigated. To evaluate therapeutic efficacy and determine the potential immunopathology associated with TERT-based ACT, we generated and analyzed the activity of TERT-specific CD8 ϩ T cells. Methods Mice and cell linesC57BL/6 mice were from Charles River Laboratories Inc. Transgenic adenocarcinoma mouse prostate (TRAMP) mice, a gift from N. M. Greenberg (Fred Hutchinson Cancer Research Center), were maintained in the mouse facility of Istituto Oncologico Veneto. Heterozygous TRAMP used in the experiments were obtained and screened as described. 10 C57BL/6-CD45.1 ϩ mice were a gift from M. P. Colombo (National Institutes of Tumors), pmel-1 TCR transgenic mice were a gift from N. Restifo (National Institutes of Health), and human leukocyte antigen (HLA)-A*0201-transgenic (HHD) mice were obtained by F. A. Lemonnier (Institut Pasteur) and maintained at Istituto di Ricerca di Biologia Molecolare. Rag2 Ϫ/Ϫ ␥ c Ϫ/Ϫ were purchased form Taconic. Mice were treated in accordance with European guidelines (http://ec.europa.eu/environment/chemicals/ lab_animals/legislation_en.htm). B16 melanoma cells were provided by N. Restifo (NIH). B16-B7.1 cells, genetically modified to express mouse B7.1 (provided by P. Della Bona, Istituto Scientifico San Raffaele) were cultured in the An Inside Blood analysis of this article appears at the front of this issue.The online version of thi...
Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients. ' 2007 Wiley-Liss, Inc.
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