RESUMO
Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Neoplasias Pulmonares/imunologia , Plasmídeos/imunologia , Receptor Toll-Like 9/imunologia , Vacinas de DNA/imunologia , Animais , Modelos Animais de Doenças , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/genética , Células Tumorais Cultivadas , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
In the last decade, immunotherapy with monoclonal antibodies targeting immunological check points has become a breakthrough therapeutic modality for solid cancers. However, only up to 50 % of patients benefit from this powerful approach. For others vaccination might provide a plausible addition or alternative. For induction of effective anticancer immunity CD4+ T cell help is required, which is often difficult to induce to self cancer targets because of tolerogenic mechanisms. Our approach for cancer vaccines has been to incorporate into the vaccine design sequences able to activate foreign T cell help, through genetically linking cancer targets to microbial sequences (King et al. in Nat Med 4(11):1281-1286, 1998; Savelyeva et al. in Nat Biotechnol 19(8):760-764, 2001). This harnesses the non-tolerized CD4 T cell repertoire available in patients to help induction of effective immunity against fused cancer antigens. Multiple immune effector mechanisms including antibody, CD8+ T cells as well as CD4 effector T cells can be activated using this strategy. Delivery via DNA vaccines has already indicated clinical efficacy. The same principle of linked T cell help has now been transferred to other novel vaccine modalities to further potentiate immunity against cancer targets.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Neoplasias/imunologia , Neoplasias/terapia , Vacinação , Linfócitos T CD8-Positivos/imunologia , HumanosRESUMO
For long-term attack on tumor cells in patients with prostate cancer, induction of cytolytic T cells is desirable. Several lineage-specific target proteins are known and algorithms have identified candidate MHC class I-binding peptides, particularly for HLA-A*0201. We have designed tolerance-breaking DNA fusion vaccines incorporating a domain of tetanus toxin fused to candidate tumor-derived peptide sequences. Using three separate peptide sequences from prostate-specific membrane antigen (PSMA) (peptides PSMA(27) , PSMA(663) , and PSMA(711) ), this vaccine design induced high levels of CD8(+) T cells against each peptide in a HLA-A(*) 0201 preclinical model. In contrast, the full-length PSMA sequence containing all three epitopes was poorly immunogenic. Induced T cells were cytotoxic against peptide-loaded tumor cells, but only those against PSMA(27) or PSMA(663) peptides, and not PSMA(711) , were able to kill tumor cells expressing endogenous PSMA. Cytotoxicity was also evident in vivo. The preclinical model provides a powerful tool for generating CD8(+) T cells able to predict whether target cells can process and present peptides, essential for planning peptide vaccine-based clinical trials.
Assuntos
Antígenos de Superfície/imunologia , Vacinas Anticâncer/imunologia , Glutamato Carboxipeptidase II/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Superfície/química , Antígenos de Superfície/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/imunologia , Glutamato Carboxipeptidase II/química , Glutamato Carboxipeptidase II/genética , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/metabolismo , Toxina Tetânica/genética , Toxina Tetânica/imunologia , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.
Assuntos
Adenocarcinoma/tratamento farmacológico , Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Colorretais/química , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Bucais/química , Miofibroblastos/patologia , NADPH Oxidases/antagonistas & inibidores , Neoplasias Orofaríngeas/química , Actinas/análise , Adenocarcinoma/química , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fibroblastos Associados a Câncer/química , Fibroblastos Associados a Câncer/fisiologia , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/genética , Contagem de Células , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Neoplasias Esofágicas/química , Neoplasias Esofágicas/genética , Feminino , Neoplasias de Cabeça e Pescoço/química , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Miofibroblastos/química , NADPH Oxidase 4 , NADPH Oxidases/análise , NADPH Oxidases/genética , Transplante de Neoplasias , Neoplasias Orofaríngeas/patologia , Fenótipo , Pirazóis/uso terapêutico , Pirazolonas , Piridinas/uso terapêutico , Piridonas , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Taxa de Sobrevida , Regulação para CimaRESUMO
Passive antibody therapy for cancer is an effective but costly treatment modality. Induction of therapeutically potent anticancer antibodies by active vaccination is an attractive alternative but has proven challenging in cancer due to tolerogenic pressure in patients. Here, we used the clinically relevant cancer target Her2, known to be susceptible to targeting by antibody therapy, to demonstrate how potent antibody can be induced by vaccination. A novel 44kD Her2 protein fragment was generated and found to be highly effective at inducing anti-Her2 antibody including trastuzumab-like reactivities. In the tolerant and spontaneous BALB-neuT mouse model of metastatic breast cancer this Her2-targeting vaccine was only effective if the fragment was conjugated to a foreign immunogenic carrier; Fragment C of tetanus toxin. Only the conjugate vaccine induced high affinity anti-Her2 antibody of multiple isotypes and suppressed tumor development. The magnitude of CD4(+) T-cell help and breadth of cytokines secreted by the CD4(+) T helper (Th) cells induced to the foreign antigen was critical. We used a highly efficient plant-based bio-manufacturing process for protein antigens, magnICON, for vaccine expression, to underpin feasibility of future clinical testing. Hence, our novel Her2-targeting conjugate vaccine combines preclinical efficacy with clinical deliverability, thus setting the scene for therapeutic testing.
RESUMO
Antibodies that block T-cell-regulatory checkpoints have recently emerged as a transformative approach to cancer treatment. However, the clinical efficacy of checkpoint blockade depends upon inherent tumor immunogenicity, with variation in infiltrating T cells contributing to differences in objective response rates. Here, we sought to understand the molecular correlates of tumor-infiltrating T lymphocytes (TIL) in squamous cell carcinoma (SCC), using a systems biologic approach to integrate publicly available omics datasets with histopathologic features. We provide evidence that links TIL abundance and therapeutic outcome to the regulation of tumor glycolysis by EGFR and HIF, both of which are attractive molecular targets for use in combination with immunotherapeutics. Cancer Res; 76(14); 4136-48. ©2016 AACR.