A TLR9 agonist enhances therapeutic effects of telomerase genetic vaccine.

The telomerase reverse transcriptase (TERT) is an attractive target for cancer vaccination because its expression is reactivated in most tumors. In this study, we have evaluated the ability of a genetic vaccine targeting murine TERT (mTERT) based on DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to exert therapeutic effects in combination with a novel TLR9 agonist, referred to as immune modulatory oligonucleotide (IMO), as an adjuvant. IMO was administered to mice at the same time as vaccine. IMO induced dose-dependent cytokine secretion and activation of NK cells. Most importantly, vaccination of mice with IMO in combination with mTERT vaccine conferred therapeutic benefit in tumor bearing animals and this effect was associated with increased NK, DC and T cell tumor infiltration. These data show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against TERT with IMO induces multiple effects on innate and adaptive immune responses resulting in a significant antitumor efficacy.

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.

In vivo administration of artificial antigen-presenting cells activates low-avidity T cells for treatment of cancer.

The development of effective antitumor immune responses is normally constrained by low-avidity, tumor-specific CTLs that are unable to eradicate the tumor. Strategies to rescue antitumor activity of low-avidity melanoma-specific CTLs in vivo may improve immunotherapy efficacy. To boost the in vivo effectiveness of low-avidity CTLs, we immunized mice bearing lung melanoma metastases with artificial antigen-presenting cells (aAPC), made by covalently coupling (pep)MHC-Ig dimers and B7.1-Ig molecules to magnetic beads. aAPC treatment induced significant tumor reduction in a mouse telomerase antigen system, and complete tumor eradication in a mouse TRP-2 antigen system, when low-avidity CTLs specific for these antigens were adoptively transferred. In addition, in an in vivo treatment model of subcutaneous melanoma, aAPC injection also augmented the activity of adoptively transferred CTLs and significantly delayed tumor growth. In vivo tumor clearance due to aAPC administration correlated with in situ proliferation of the transferred CTL. In vitro studies showed that aAPC effectively stimulated cytokine release, enhanced CTL-mediated lysis, and TCR downregulation in low-avidity CTLs. Therefore, in vivo aAPC administration represents a potentially novel approach to improve cancer immunotherapy.

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.

Immunological markers of cancer vaccine efficacy and their clinical relevance.

As the field of anticancer therapy advances, with a mission to either prevent tumors or improve the quality of life and survival of those affected by the disease, biomarker discovery in oncology is also growing. Since cancer therapy has become a type of personalized medicine, biomarkers are strongly needed in order to identify the right therapy for the right patient and predict efficacy in a timely manner so that alternative approaches can be tested. In this article, the author summarizes the most recent studies published on cancer vaccines where immunological parameters were analyzed to assess immunogenicity and, most importantly, to establish correlations with clinical benefit. As highlighted here, the complexity of both the disease and the immune system still makes it a very challenging discovery process.

Preventive vaccination with telomerase controls tumor growth in genetically engineered and carcinogen-induced mouse models of cancer.

The telomerase reverse transcriptase, TERT, is an attractive target for human cancer vaccination because its expression is reactivated in a conspicuous fraction of human tumors. Genetic vaccination with murine telomerase (mTERT) could break immune tolerance in different mouse strains and resulted in the induction of both CD4+ and CD8+ telomerase-specific T cells. The mTERT-derived immunodominant epitopes recognized by CD8+ T cells were further defined in these mouse strains and used to track immune responses. Antitumor efficacy of telomerase-based vaccination was investigated in two cancer models closely resembling human diseases: the TRAMP transgenic mice for prostate cancer and a carcinogen-induced model for colon cancer. TERT overexpression in tumor lesions was shown in both models by immunohistochemistry, thus reinforcing the similarity of these tumors to their human counterparts. Repeated immunizations with mTERT-encoding DNA resulted in a significant delay of tumor formation and progression in both the prostate cancer and the colon cancer models. Moreover, evaluation of the intratumoral infiltrate revealed the presence of telomerase-specific T cells in vaccinated mice. The safety of vaccination was confirmed by the absence of histomorphologic changes on postnecropsy analysis of several organs and lack of adverse effects on blood cell counts. These results indicate that TERT vaccination can elicit antigen-specific immunosurveillance and imply this antigen as a potential candidate for preventive cancer vaccines.

Therapeutic vaccination halts disease progression in BALB-neuT mice: the amplitude of elicited immune response is predictive of vaccine efficacy.

The aim of this study was to evaluate the efficacy of genetic vaccination with rat ErbB2 antigen in a therapeutic setting for the BALB-neuT mouse model of mammary carcinoma and to establish immunological correlates with vaccine efficacy. To define an early therapeutic setting we performed imaging studies of mouse mammary glands with a high-frequency ultrasound system that allowed the diagnosis of tumor lesions before they become palpable, starting from week 13 after mouse births. An intensive immunization protocol of vaccination was implemented at this stage, consisting of four weekly DNA injections with electroporation followed by two injections of adenovirus carrying the codon usage-optimized cDNA encoding the extracellular-transmembrane domain of rat ErbB2. Immunological parameters were monitored in each individual mouse by analyzing peripheral blood leukocytes. The appearance of the first palpable tumor in vaccinated mice was delayed and there was a statistically significant time gap before additional masses developed, indicating disease stabilization. As a result of the immunization, antibodies and CD8(+) T cells to rat ErbB2 were detected and the amplitude of elicited responses correlated with the efficacy of vaccination. Moreover, the vaccination regimen specifically halted the rise in circulating myeloid suppressor cells (MSCs). All three parameters, that is, CD8(+) T cells, antibodies to rat ErbB2, and circulating MSCs, measured at the end of vaccination could be used as predictive biomarkers for future tumor development. This study emphasizes the potential of genetic vaccines for the therapeutic treatment of malignancies and suggests possible predictive biomarkers to be further validated in the clinic for the follow-up of vaccinated cancer patients.

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.

Adenovirus vaccination against neu oncogene exerts long-term protection from tumorigenesis in BALB/neuT transgenic mice.

The transforming rat HER2/neu oncogene (neu), when embedded in the genome of transgenic BALB/c (neuT) mice, provokes the development of an invasive carcinoma in each of their 10 mammary glands. We used the neuT mice model system to evaluate the immunization efficiency and the protective effect of intramuscular injection of adenovirus (Ad) and/or of DNA with electrostimulation (DNA+ES), both expressing the rat p185(neu) protein. A neu cDNA sequence, which exclusively contains codons preferred by highly expressed mammalian genes, was used in this study. This "optimized" cDNA displayed higher expression in cultured cells and greater cell-mediated response than the original gene when injected as DNA+ES. Ad expressing the optimized sequence (Ad5-neu.opt) induced a higher immune response, as measured by the frequency of IFN-gamma-secreting spleen cells and antibody titers. Different Ad/DNA combinations and immunization schedules confirmed the superiority of Ad5-neu.opt in inducing a strong Th1-skewed humoral and CD8(+) cell-mediated response. Two Ad5-neu.opt injections of 10(9) viral particles at week 10 and 12 were sufficient to induce the highest response, which persisted at detectable levels up to 33 weeks of age. Anti-Ad5 antibodies elicited by previous injections neutralized the effect of an additional Ad5-neu.opt immunization at week 19. A group, which received 3 injections of DNA+ES at week 23, 27 and 31, in addition to the 3 Ad injections at week 10, 12 and 19 showed an increased frequency of IFN-gamma(+), CD8(+) PBMC at week 25, which persisted at detectable levels till week 38. Ad5-neu.opt administration at 10 and 12 weeks of age had a significant impact on tumor progression. At 44 weeks, 40% of the mice were completely protected from tumors with a mean tumor of 3.8. In contrast, control mice developed 10 tumors and died by week 27. Vaccination blocked the tumor development at the atypical hyperplasia stage present at the time of treatment. Tumors developing at later times express reduced levels of rat p185(neu) protein.

Upregulation of group 1 CD1 antigen presenting molecules in guinea pigs with experimental autoimmune encephalomyelitis: an immunohistochemical study.

In humans, group 1 CD1 glycoproteins present foreign and self lipid and glycolipid antigens to T-cells. Homologues of these molecules are not found in mice or rats but are present in guinea pigs (GPs). We examined CD1 and MHC class II expression in the central nervous system (CNS) of GPs sensitized for experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. In normal GPs and the uninflamed CNS, low-level MHC class II (MHC II) immunoreactivity occurred on vascular elements, meningeal macrophages and parenchymal microglial cells, whereas immunoreactivity for CD1 was absent. In the inflamed CNS, the majority of infiltrating cells were MHC II+ and microglia showed increased expression. CD1 immunoreactivity was detected on astrocytes and subsets of inflammatory cells Including B cells and macrophages. Minimal CD1 and MHC II co-expression was noted on inflammatory cells or glia. We conclude that group 1 CD1 molecules are strongly upregulated in the inflamed CNS on subsets of cells distinct from the majority of MHC II bearing cells. The expression of CD1 proteins in such lesions broadens the potential repertoire of antigens recognized at these sites and highlights the value of the GP as a model for studies of the relevance of CD1 molecules in host defense and autoimmune diseases.

Involvement of classical and novel protein kinase C isoforms in the response of human V gamma 9V delta 2 T cells to phosphate antigens.

Human gammadelta T cells expressing the Vgamma9Vdelta2 gene segments are activated polyclonally by phosphoantigens found on a wide variety of pathogenic organisms. After ligand exposure, Vgamma9Vdelta2 T cells proliferate and rapidly secrete large amounts of cytokines and chemokines that contribute to the innate immune response to these pathogens. Neither APCs nor costimulatory molecules are required. In this study we examined whether these phosphoantigens activate protein kinase Ctheta (PKCtheta). This novel PKC isoform is essential for Ag signaling through the alphabeta TCR in a costimulation-dependent fashion. The results showed that isopentenyl pyrophosphate (IPP), a soluble phospholigand released by mycobacteria, led to the rapid and persistent activation of PKCtheta in gammadelta T cells, as determined by evidence of translocation and phosphorylation. In contrast, no ligand-dependent response was detected for PKCalpha/beta or PKCdelta. Using the inhibitors Gö6976 and rottlerin, a role for both conventional and novel PKC isoforms in IPP-induced proliferation, CD25 expression, and cytokine and chemokine production was demonstrated. Gel-shift assays indicated that the transcription factors NF-kappaB and AP-1 were downstream targets of PKC activation. IPP also induced the rapid and persistent phosphorylation of extracellular signal-regulated kinases 1 and 2, p38 mitogen-activated kinase, and stress-activated kinase/c-Jun N-terminal kinase, but only an inhibitor of conventional PKCs blocked these responses. We conclude that the gammadelta T cell response to phosphoantigens is regulated by both novel and conventional PKC isoforms, with PKCtheta being more responsive to ligand stimulation and PKCalpha/beta to growth-factor availability.

Granulocyte-macrophage colony-stimulating factor induces an expression program in neonatal microglia that primes them for antigen presentation.

Neonatal microglial cells respond to GM-CSF and M-CSF by acquiring different morphologies and phenotypes. To investigate the extent and consequences of this process, a global gene expression analysis was performed, with significant changes in transcript levels confirmed by biochemical analyses. Primary murine microglial cells underwent substantial expression reprogramming after treatment with GM-CSF or M-CSF with many differentially expressed transcripts important in innate and adaptive immunity. In particular, many gene products involved in Ag presentation were induced by GM-CSF, but not M-CSF, thus potentially priming relatively quiescent microglia cells for Ag presentation. This function of GM-CSF is distinct from its primary function in cell proliferation and survival.