Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors
. For treatments using checkpoint inhibitors to be ...successful, a high mutational load and responses to neoepitopes are thought to be essential
. There is limited intratumoural infiltration of immune cells
in glioblastoma and these tumours contain only 30-50 non-synonymous mutations
. 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.
Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial ...mouse tumor, the CT26 colon carcinoma cell line.
We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class II is expressed. Several known cancer-testis antigens are expressed, including Atad2, Cep55, and Pbk. The highest expressed gene is a mutated form of the mouse tumor antigen gp70. Of the 1,688 non-synonymous point variations, 154 are both in expressed genes and in peptides predicted to bind MHC and thus potential targets for immunotherapy development. Based on its molecular signature, we predicted that CT26 is refractory to anti-EGFR mAbs and sensitive to MEK and MET inhibitors, as have been previously reported.
CT26 cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. As CT26 is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutation-derived neoantigens distinguish tumor from normal cells. T cells can sense the HLA-presented mutations, recognize tumor cells as non-self and destroy them. Therapeutically, immunotherapy ...antibodies can increase the virulence of the immune system by increasing T-cell cytotoxicity targeted toward neoantigens. Neoantigen vaccines act through antigen-presenting cells, such as dendritic cells, to activate patient-endogenous T cells that recognize vaccine-encoded mutations. Infusion of mutation-targeting T cells by adoptive cell therapy (ACT) directly increases the number and frequency of cytotoxic T cells recognizing and killing tumor cells. At the same time, publicly-funded consortia have profiled tumor genomes across many indications, identifying mutations in each tumor. For example, we find basal and HER2 positive tumors contain more mutated proteins and more TP53 mutations than luminal A/B breast tumors. HPV negative tumors have more mutated proteins than HPV positive head and neck tumors and in agreement with the hypothesis that HPV activity interferes with p53 activity, only 14% of the HPV positive mutations have TP53 mutations vs. 86% of the HPV negative tumors. Lung adenocarcinomas in smokers have over four times more mutated proteins relative to those in never smokers (median 248 vs. 61, respectively). With an eye toward immunotherapy applications, we review the spectrum of mutations in multiple indications, show variations in indication sub-types, and examine intra- and inter-indication prevalence of re-occurring mutation neoantigens that could be used for warehouse vaccines and ACT.
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.
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'.
Angiosarcoma is an uncommon endothelial malignancy and a highly aggressive soft tissue sarcoma. Due to its infiltrative nature, successful management of localized angiosarcoma is often challenging. ...Systemic chemotherapy is used in the metastatic setting and occasionally in patients with high-risk localized disease in neoadjuvant or adjuvant settings. However, responses tend to be short-lived and most patients succumb to metastatic disease. Novel therapies are needed for patients with angiosarcomas.
We performed a retrospective analysis of patients with locally advanced or metastatic angiosarcoma, who were treated with checkpoint inhibitors at our institution. We collected their clinical information and outcome measurements. In one patient with achieved complete response, we analyzed circulating and infiltrating T cells within peripheral blood and tumor tissue.
We have treated seven angiosarcoma (AS) patients with checkpoint inhibitors either in the context of clinical trials or off label Pembrolizumab + Axitinib (NCT02636725; n = 1), AGEN1884, a CTLA-4 inhibitor (NCT02694822; n = 2), Pembrolizumab (n = 4). Five patients had cutaneous angiosarcoma, one primary breast angiosarcoma and one radiation-associated breast angiosarcoma. At 12 weeks, 5/7 patients (71%) had partial response of their lesions either on imaging and/or clinical exam and two (29%) had progressive disease. 6/7 patients are alive to date and, thus far, 3/7 patients (43%) have progressed (median 3.4 months)- one achieved partial response after pembrolizumab was switched to ongoing Nivolumab/Ipilimumab, one died of progressive disease at 31 weeks (primary breast angiosarcoma) and one was placed on pazopanib. One patient had a complete response (CR) following extended treatment with monotherapy AGEN1884. No patient experienced any ≥ grade 2 toxicities.
This case series underscores the value of targeted immunotherapy in treating angiosarcoma. It also identifies genetic heterogeneity of cutaneous angiosarcomas and discusses specific genetic findings that may explain reported benefits from immunotherapy.
Rates of SNPs (single nucleotide polymorphisms) and cross-species genomic sequence conservation reflect intra- and inter-species variation, respectively. Here, I report SNP rates and genomic sequence ...conservation adjacent to mRNA processing regions and show that, as expected, more SNPs occur in less conserved regions and that functional regions have fewer SNPs. Results are confirmed using both mouse and human data. Regions include protein start codons, 3' splice sites, 5' splice sites, protein stop codons, predicted miRNA binding sites, and polyadenylation sites. Throughout, SNP rates are lower and conservation is higher at regulatory sites. Within coding regions, SNP rates are highest and conservation is lowest at codon position three and the fewest SNPs are found at codon position two, reflecting codon degeneracy for amino acid encoding. Exon splice sites show high conservation and very low SNP rates, reflecting both splicing signals and protein coding. Relaxed constraint on the codon third position is dramatically seen when separating exonic SNP rates based on intron phase. At polyadenylation sites, a peak of conservation and low SNP rate occurs from 30 to 17 nt preceding the site. This region is highly enriched for the sequence AAUAAA, reflecting the location of the conserved polyA signal. miRNA 3' UTR target sites are predicted incorporating interspecies genomic sequence conservation; SNP rates are low in these sites, again showing fewer SNPs in conserved regions. Together, these results confirm that SNPs, reflecting recent genetic variation, occur more frequently in regions with less evolutionarily conservation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
HLA and proteasome expression body map Boegel, Sebastian; Löwer, Martin; Bukur, Thomas ...
BMC medical genomics,
03/2018, Letnik:
11, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The presentation of HLA peptide complexes to T cells is a highly regulated and tissue specific process involving multiple transcriptionally controlled cellular components. The extensive polymorphism ...of HLA genes and the complex composition of the proteasome make it difficult to map their expression profiles across tissues.
Here we applied a tailored gene quantification pipeline to 4323 publicly available RNA-Seq datasets representing 55 normal tissues and cell types to examine expression profiles of (classical and non-classical) HLA class I, class II and proteasomal genes.
We generated the first comprehensive expression atlas of antigen presenting-related genes across 56 normal tissues and cell types, including immune cells, pancreatic islets, platelets and hematopoietic stem cells. We found a surprisingly heterogeneous HLA expression pattern with up to 100-fold difference in intra-tissue median HLA abundances. Cells of the immune system and lymphatic organs expressed the highest levels of classical HLA class I (HLA-A,-B,-C), class II (HLA-DQA1,-DQB1,-DPA1,-DPB1,-DRA,-DRB1) and non-classical HLA class I (HLA-E,-F) molecules, whereas retina, brain, muscle, megakaryocytes and erythroblasts showed the lowest abundance. In contrast, we identified a distinct and highly tissue-restricted expression pattern of the non-classical class I gene HLA-G in placenta, pancreatic islets, pituitary gland and testis. While the constitutive proteasome showed relatively constant expression across all tissues, we found the immunoproteasome to be enriched in lymphatic organs and almost absent in immune privileged tissues.
Here, we not only provide a reference catalog of tissue and cell type specific HLA expression, but also highlight extremely variable expression of the basic components of antigen processing and presentation in different cell types. Our findings indicate that low expression of classical HLA class I molecules together with lack of immunoproteasome components as well as upregulation of HLA-G may be of key relevance to maintain tolerance in immune privileged tissues.
Immune checkpoint blockade (ICB) is currently evaluated in patients with glioblastoma (GBM), based on encouraging clinical data in other cancers, and results from studies with the ...methylcholanthrene-induced GL261 mouse glioma. In this paper, we describe a novel model faithfully recapitulating some key human GBM characteristics, including low mutational load, a factor reported as a prognostic indicator of ICB response. Consistent with this observation, SB28 is completely resistant to ICB, contrasting with treatment sensitivity of the more highly mutated GL261. Moreover, SB28 shows features of a poorly immunogenic tumor, with low MHC-I expression and modest CD8
T-cell infiltration, suggesting that it may present similar challenges for immunotherapy as human GBM. Based on these key features for immune reactivity, SB28 may represent a treatment-resistant malignancy likely to mirror responses of many human tumors. We therefore propose that SB28 is a particularly suitable model for optimization of GBM immunotherapy.
From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. ...More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated > 10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development.
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