Adoptive transfer of dendritic cells (DCs) transfected with in vitro–transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer ...immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3′ of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription–polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3′ end rather than one extended with unrelated nucleotides, and 2 sequential β-globin 3′ untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4+ and CD8+ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.
Purpose: Antibody-based cancer therapies have emerged as the most promising therapeutics in oncology. The purpose of this study was
to discover novel targets for therapeutic antibodies in solid ...cancer.
Experimental Design: We combined data mining and wet-bench experiments to identify strictly gastrocyte lineage–specific cell surface molecules
and to validate them as therapeutic antibody targets.
Results: We identified isoform 2 of the tight junction molecule claudin-18 (CLDN18.2) as a highly selective cell lineage marker. Its
expression in normal tissues is strictly confined to differentiated epithelial cells of the gastric mucosa, but it is absent
from the gastric stem cell zone. CLDN18.2 is retained on malignant transformation and is expressed in a significant proportion
of primary gastric cancers and the metastases thereof. In addition to its orthotopic expression, we found frequent ectopic
activation of CLDN18.2 in pancreatic, esophageal, ovarian, and lung tumors, correlating with distinct histologic subtypes.
The activation of CLDN18.2 depends on the binding of the transcription factor cyclic AMP–responsive element binding protein
to its unmethylated consensus site. Most importantly, we were able to raise monoclonal antibodies that bind to CLDN18.2 but
not to its lung-specific splice variant and recognize the antigen on the surface of cancer cells.
Conclusions: Its highly restricted expression pattern in normal tissues, its frequent ectopic activation in a diversity of human cancers,
and the ability to specifically target this molecule at the cell surface of tumor cells qualify CLDN18.2 as a novel, highly
attractive pan-cancer target for the antibody therapy of epithelial tumors.
Cancer is characterized by an accumulation of genetic alterations. Somatic mutations can generate cancer-specific neoepitopes that are recognized by autologous T cells as foreign and constitute ideal ...cancer vaccine targets. Every tumor has its own unique composition of mutations, with only a small fraction shared between patients. Technological advances in genomics, data science, and cancer immunotherapy now enable the rapid mapping of the mutations within a genome, rational selection of vaccine targets, and on-demand production of a therapy customized to a patient's individual tumor. First-in-human clinical trials of personalized cancer vaccines have shown the feasibility, safety, and immunotherapeutic activity of targeting individual tumor mutation signatures. With vaccination development being promoted by emerging innovations of the digital age, vaccinating a patient with individual tumor mutations may become the first truly personalized treatment for cancer.
Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. ...Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4(+) T cells. Vaccination with such CD4(+) immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4(+) T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal ...heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.
Although naked antigen-encoding RNA has entered clinical testing, basic knowledge on how to apply this promising novel vaccine format is still pending. By comparing different administration routes, ...we observed surprisingly potent antigen-specific T-cell immunity upon intranodal injection of naked antigen-encoding RNA. RNA was selectively uptaken by resident dendritic cells, propagated a T-cell attracting and stimulatory intralymphatic milieu, and led to efficient expansion of antigen-specific CD8+ as well as CD4+ T cells. By intranodal treatment of mice with repeated cycles of RNA, we achieved de novo priming of naïve T cells, which became potent cytolytic effectors capable of homing to primary and secondary lymphatic tissues as well as memory T cells. In tumor-bearing mice intralymphatic RNA vaccination elicited protective and therapeutic antitumor immune responses, resulting in a remarkable survival benefit as compared with other treatment regimens. This is the first report of strong systemic antigen-specific Th1-type immunity and cancer cure achieved with naked antigen-encoding RNA in preclinical animal models.
In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by ...structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Claudin-18 isoform 2 (CLDN18.2) is one of the few members of the human claudin family of tight junction molecules with strict restriction to one cell lineage. The objective of the current study was ...to compare molecular structure and tissue distribution of this gastrocyte specific molecule in mammals. We show here that the CLDN18.2 protein sequence is highly conserved, in particular with regard to functionally relevant domains in mouse, rat, rabbit, dog, monkey and human and also in lizards. Moreover, promoter regions of orthologs are highly homologous, including the binding site of the transcription factor cyclic AMP–responsive element binding protein (CREB), which is known to regulate activation of human CLDN18.2. Employing RT-PCR and immunohistochemistry, we found that, analogous to the human gene, all orthologous CLDN18.2 transcripts and proteins are exclusively expressed in differentiated gastric cells. Gene structure, promoter elements and RNA expression pattern of the lung-tissue specific Claudin-18 isoform 1 (CLDN18.1) as well, are homologous across species. These findings exemplify phylogenetic conservation of lineage-specific members of a multigene family. Given that CLDN18.2 is a novel drug target candidate, our data is also relevant for drug development as it reveals all six investigated mammalian species as suitable models for testing safety of CLDN18.2 targeting regimen.
We present a method, seq2HLA, for obtaining an individual's human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are ...mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and requires no change to laboratory protocols, it can be used for both existing datasets and future studies, thus adding a new dimension for HLA typing and biomarker studies.
The ability to control autoreactive T cells without inducing systemic immune suppression is the major goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery ...of pharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Ψ mRNA) coding for disease-related autoantigens results in antigen presentation on splenic CD11c
antigen-presenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis, the disease is suppressed by treatment with such m1Ψ mRNA. The treatment effect is associated with a reduction of effector T cells and the development of regulatory T cell (T
cell) populations. Notably, these T
cells execute strong bystander immunosuppression and thus improve disease induced by cognate and noncognate autoantigens.
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