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1.
Recent Results Cancer Res ; 214: 153-167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31473852

RESUMO

After more than a century of efforts to establish cancer immunotherapy in clinical practice, the advent of checkpoint inhibition (CPI) therapy was a critical breakthrough toward this direction (Hodi et al. in Cell Rep 13(2):412-424, 2010; Wolchok et al. in N Engl J Med 369(2):122-133, 2013; Herbst et al. in Nature 515(7528):563-567, 2014; Tumeh et al. in Nature 515(7528):568-571, 2014). Further, CPIs shifted the focus from long studied shared tumor-associated antigens to mutated ones. As cancer is caused by mutations in somatic cells, the concept to utilize these correlates of 'foreignness' to enable recognition and lysis of the cancer cell by T cell immunity seems an obvious thing to do.


Assuntos
Vacinas Anticâncer , Epitopos/imunologia , Imunoterapia , Neoplasias/terapia , Antígenos de Neoplasias/imunologia , Humanos
3.
Mol Cell Proteomics ; 18(6): 1255-1268, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31154438

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

5.
Nature ; 565(7738): 240-245, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30568303

RESUMO

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.


Assuntos
Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Glioblastoma/diagnóstico , Glioblastoma/terapia , Medicina de Precisão/métodos , Adulto , Idoso , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Glioblastoma/imunologia , Antígenos HLA-A/imunologia , Humanos , Memória Imunológica/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Auxiliares-Indutores/imunologia , Resultado do Tratamento
6.
Mol Cell Proteomics ; 17(11): 2132-2145, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30072578

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

7.
Hum Gene Ther ; 28(12): 1138-1146, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28877647

RESUMO

Among nucleic acid-based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-ß upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.


Assuntos
Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Evasão da Resposta Imune , RNA , Vírus Vaccinia/genética , Proteínas Virais , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA/genética , RNA/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genética , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
8.
Nature ; 547(7662): 222-226, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28678784

RESUMO

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.


Assuntos
Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Melanoma/imunologia , Melanoma/terapia , Mutação/genética , Medicina de Precisão/métodos , RNA/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/imunologia , Antígenos CD8/imunologia , Vacinas Anticâncer/uso terapêutico , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoterapia/métodos , Melanoma/genética , Metástase Neoplásica , Recidiva Local de Neoplasia/prevenção & controle , Nivolumabe , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/imunologia , Vacinação , Microglobulina beta-2/deficiência
9.
Methods Mol Biol ; 1499: 203-222, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27987152

RESUMO

A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist that specifically address mRNA-based vaccines. The existing regulatory framework, however, clearly defines that mRNA-based vaccines in most cases have to be centrally approved. Interestingly, depending on whether RNA-based vaccines are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively. Besides an overview on the current clinical use of mRNA vaccines in various therapeutic areas a detailed discussion of the current regulatory situation is provided and regulatory perspectives are discussed.


Assuntos
Vacinas Anticâncer/imunologia , RNA Mensageiro/imunologia , Animais , Antígenos/imunologia , Europa (Continente) , Terapia Genética/métodos , Humanos , Neoplasias/imunologia , Neoplasias/terapia
10.
Methods Mol Biol ; 1499: 223-236, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27987153

RESUMO

Cancer accumulates 10s to 1000s of genomic mutations of which a fraction is immunogenic and may serve as an Achilles' heel of tumor cells. Mutation-specific T cells can recognize these antigens and destroy malignant cells. Strategies to immunotherapeutically address individual tumor mutations employing peptide or mRNA based vaccines are now actively investigated in mice and humans. An important step of determining the therapeutic potential of a mutanome vaccine is the detection of mutation reactive T-cell responses. In this chapter we provide protocols to identify and subtype mutation specific T cells in mice based on IFN-γ ELISpot and flow cytometry.


Assuntos
Epitopos de Linfócito T/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Humanos , Imunoterapia/métodos , Interferon gama/imunologia , Camundongos , Mutação/imunologia
11.
Curr Issues Mol Biol ; 22: 113-128, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27801664

RESUMO

mRNA vaccines are finally ready to assume their rightful place at the forefront of nucleic acid- based vaccines. Major achievements within the last two decades have turned this highly versatile molecule into a safe and very attractive pharmaceutical platform that combines many positive attributes able to address a broad range of diseases, including cancer. The simplicity of mRNA vaccines greatly reduces complications generally associated with the production of biological vaccines. Intrinsic costimulatory and inflammatory triggers in addition to the provision of the antigenic information makes mRNA an all- in-one molecule that does not need additional adjuvants and that does not pose the risk of genomic integration. Clinical studies in various cancer types are moving forward and promising results with favorable clinical outcome are awaited. This review will recapitulate conceptual, mechanistic and immune-related features of this highly versatile molecule, elucidate how these features have been addressed in the past, and how comprehensive understanding can foster further optimization for broad application possibilities in cancer treatment.


Assuntos
Vacinas Anticâncer/imunologia , Neoplasias/terapia , RNA Mensageiro/genética , Vacinas Anticâncer/genética , Humanos , Imunoterapia Ativa/normas , Imunoterapia Ativa/tendências , RNA Mensageiro/imunologia
14.
Cancer Immunol Immunother ; 65(9): 1075-83, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27422115

RESUMO

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose-response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30 days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8(+) T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/metabolismo , RNA/farmacocinética , Animais , Células Dendríticas/imunologia , Feminino , Humanos , Injeções Intradérmicas , Camundongos , Camundongos Endogâmicos C57BL , Pinocitose , RNA/administração & dosagem
15.
Nature ; 534(7607): 396-401, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27281205

RESUMO

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunoterapia/métodos , Melanoma/imunologia , Melanoma/terapia , RNA/administração & dosagem , Administração Intravenosa , Animais , Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/genética , Antígenos Virais/genética , Autoantígenos/genética , Autoantígenos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Ensaios Clínicos Fase I como Assunto , Células Dendríticas/citologia , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Feminino , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , RNA/genética , Eletricidade Estática , Linfócitos T/citologia , Linfócitos T/imunologia , Receptor 7 Toll-Like/imunologia
16.
Methods Mol Biol ; 1428: 163-75, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27236799

RESUMO

Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Células da Medula Óssea/citologia , Proteínas de Membrana/administração & dosagem , RNA Mensageiro/administração & dosagem , Animais , Células da Medula Óssea/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/imunologia , Vacinação/métodos
17.
Clin Cancer Res ; 22(8): 1885-96, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27084742

RESUMO

Somatic mutations binding to the patient's MHC and recognized by autologous T cells (neoepitopes) are ideal cancer vaccine targets. They combine a favorable safety profile due to a lack of expression in healthy tissues with a high likelihood of immunogenicity, as T cells recognizing neoepitopes are not shaped by central immune tolerance. Proteins mutated in cancer (neoantigens) shared by patients have been explored as vaccine targets for many years. Shared ("public") mutations, however, are rare, as the vast majority of cancer mutations in a given tumor are unique for the individual patient. Recently, the novel concept of truly individualized cancer vaccination emerged, which exploits the vast source of patient-specific "private" mutations. Concurrence of scientific advances and technological breakthroughs enables the rapid, cost-efficient, and comprehensive mapping of the "mutanome," which is the entirety of somatic mutations in an individual tumor, and the rational selection of neoepitopes. How to transform tumor mutanome data to actionable knowledge for tailoring individualized vaccines "on demand" has become a novel research field with paradigm-shifting potential. This review gives an overview with particular focus on the clinical development of such vaccines.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Variação Genética/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Variação Antigênica , Antígenos de Neoplasias/genética , Vacinas Anticâncer/administração & dosagem , Ensaios Clínicos como Assunto , Epitopos/genética , Heterogeneidade Genética , Humanos , Mutação , Neoplasias/genética , Pesquisa Médica Translacional
20.
Curr Opin Immunol ; 39: 14-22, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26716729

RESUMO

Somatic mutations are important drivers of cancer development. Accumulating evidence suggests that a significant subset of mutations result in neo-epitopes recognized by autologous T cells and thus may constitute the Achilles' heel of tumor cells. T cells directed against mutations have been shown to have a key role in clinical efficacy of potent cancer immunotherapy modalities, such as adoptive transfer of autologous tumor infiltrating lymphocytes and immune checkpoint inhibitors. Whereas these findings strengthen the idea of a prominent role of neo-epitopes in tumor rejection, the systematic therapeutic exploitation of mutations was hampered until recently by the uniqueness of the repertoire of mutations ('the mutanome') in every patient's tumor. This review highlights insights into immune recognition of neo-epitopes and novel concepts for comprehensive identification and immunotherapeutic exploitation of individual mutations.


Assuntos
Antígenos de Neoplasias/genética , Imunoterapia , Mutação/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Humanos , Neoplasias/imunologia
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