Autoimmune B-cell lymphopenia after successful adoptive therapy with telomerase-specific T lymphocytes.

Telomerase reverse transcriptase (TERT) is a good candidate for cancer immunotherapy because it is overexpressed in 85% of all human tumors and implicated in maintenance of the transformed phenotype. TERT-based cancer vaccines have been shown to be safe, not inducing any immune-related pathology, but their impact on tumor progression is modest. Here we show that adoptive cell therapy with the use of high-avidity T lymphocytes reactive against telomerase can control the growth of different established tumors. Moreover, in transgenic adenocarcinoma mouse prostate mice, which develop prostate cancer, TERT-based adoptive cell therapy halted the progression to more aggressive and poorly differentiated tumors, significantly prolonging mouse survival. We also demonstrated that human tumors, including Burkitt lymphoma, and human cancer stem cells, are targeted in vivo by TERT-specific cytotoxic T lymphocytes. Effective therapy with T cells against telomerase, different from active vaccination, however, led to autoimmunity marked by a consistent, although transient, B-cell depletion in primary and secondary lymphoid organs, associated with alteration of the spleen cytoarchitecture. These results indicate B cells as an in vivo target of TERT-specific cytotoxic T lymphocytes during successful immunotherapy.

Coadministration of telomerase genetic vaccine and a novel TLR9 agonist in nonhuman primates.

The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.

ErbB2 genetic cancer vaccine in nonhuman primates: relevance of single nucleotide polymorphisms.

Aberrant Her2/neu expression is associated with the development of epithelial-derived human carcinomas and for this reason it is considered a good target for immunologic intervention. To define methods to circumvent immunologic tolerance and to elicit immunity against the Her2/neu tumor-associated antigen in a suitable animal model, we have isolated the cDNA encoding the rhesus monkey homolog of human Her2/neu (RhErbB2) to construct DNA plasmids and adenoviral vectors for the development of a cancer vaccine against this protein. To further increase the immunogenic potency of these vectors, a synthetic codon-optimized RhErbB2 cDNA (RhErbB2OPT) was constructed and characterized. Genetic vaccination of rhesus monkeys was effective in inducing a response against RhErbB2 in immunized animals; importantly, the elicited immunity was associated with natural RhErbB2 polymorphisms, thus distinguishing responses against "self " and "nonself " epitopes. In particular, the postpriming response recognized mainly nonself epitopes whereas the boosted response cross-reacted with self epitopes. Our findings are particularly relevant in the investigation of the impact of TAA polymorphisms on the efficacy of a cancer vaccine strategy.

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.

Genetic vaccines against Ep-CAM break tolerance to self in a limited subset of subjects: initial identification of predictive biomarkers.

The epithelial cell adhesion molecule, Ep-CAM, has been historically considered a target of passive immunotherapy using monoclonal antibodies, and more recently, of a first Pox-vector-based cancer vaccine Phase I trial in colorectal cancer patients. To shed further light on the use of this antigen, we isolated the mouse and rhesus homologues of human Ep-CAM and explored different genetic vaccination modalities based on the use of adenoviral vectors as well as DNA electroporation (DNA-EP). Immune responses to Ep-CAM were measured by IFN-gamma ELISPOT and intracellular staining assays using overlapping sets of peptides covering the entire coding regions. We found the most powerful vaccination regimen to be constituted by DNA-EP-prime/Adeno-boost mixed-modality protocols. Vaccination in rhesus macaques resulted in breakage of immunological tolerance in a minority of cases. Similarly, a low frequency of responders was observed with the mouse Ep-CAM vaccine in outbred CD1 mice. When immunized CD1 mice were analyzed for MHC haplotype and TCR expression levels, we observed that immune responders all had the same q/q MHC class I haplotype and showed higher expression levels of the TCRVbeta4 and TCRVbeta8 T cell receptors. Our results underscore the current limitations in our capacity to induce efficient cancer vaccines against self antigens like Ep-CAM, but also represent a first effort to identify predictive biomarkers of response.

Efficient induction of T-cell responses to carcinoembryonic antigen by a heterologous prime-boost regimen using DNA and adenovirus vectors carrying a codon usage optimized cDNA.

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+ and CD8+ T-cell response to the target antigen, measured by both IFNgamma-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+ and CD8+ 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+ 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.

Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates.

BACKGROUND: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates. Transduction can be improved in several animal models by application of electric pulses after DNA injection. METHODS: We have designed a modified EPO gene version by changing the EPO leader sequence and optimizing the gene codon usage. This modified gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effect. CONCLUSIONS: 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.

Hyaluronidase increases electrogene transfer efficiency in skeletal muscle.

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.