Patrizia Giannetti

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance—i.e., 100% of treated animals showing sustained expression after 4 months—was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

Immunogenicity and safety of a DNA prime/adenovirus boost vaccine against rhesus CEA in nonhuman primates

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.

Immune responses against tetracycline‐dependent transactivators affect long‐term expression of mouse erythropoietin delivered by a helper‐dependent adenoviral vector

Helper‐dependent adenoviral (HD‐Ad) vectors give rise to sustained gene expression after delivery in a variety of organisms. In particular, we previously documented persistent expression of erythropoietin (EPO) in mice after a single intramuscular (i.m.) injection of a HD‐Ad vector harboring the mouse EPO cDNA.

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4+CD25+ regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8+, CD4+ T cells and antibody response. The appearance of a CD4+ T-cell response correlated with a stronger memory response. The combined CD25+ inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

A therapeutic cancer vaccine targeting carcinoembryonic antigen in intestinal carcinomas.

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.

A Novel Mouse Model for Evaluation and Prediction of HLA-A2-restricted CEA Cancer Vaccine Responses

Human leukocyte antigen (HLA)-A2.1 transgenic mice (HHD) represent a valuable model to study and predict the immunogenicity of vaccines against pathogens. However, HHD mice are unsuitable for in vivo studies of cancer vaccines against human tumor-associated antigens because they lack T-cell tolerance that is key to define the potency of the treatment. In this study, we developed HHD/carcinoembryonic antigen P(CEA) hybrid mice by breeding transgenic mice homozygous for CEA with HHD. These mice express human CEA, present epitopes solely through HLA-A2.1 molecules and constitute a unique in vivo animal model to study HLA-A2.1-restricted immune response of a human CEA-based vaccine. Owing to the immune tolerance, HHD/CEA mice show a limited immune response and expansion of a different and restricted T-cell receptor repertoire after antigen-specific stimulation. Our data show that genetic vectors expressing CEA and peptide-based vaccines are able to efficiently break immune tolerance against CEA and to elicit strong immune response against HLA-A2.1-restricted CEA epitopes. Most importantly, efficient lysis of human CEA+/HLA-A2.1+ tumor cells was observed and significant protection against HHD/CEA tumor cells was achieved in HHD/CEA-vaccinated mice. Hence, HHD/CEA provides a relevant model for the evaluation of the potential efficacy of human CEA-based vaccines.