Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour ...tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.
Colorectal and lung cancers account for one-third of all cancer-related deaths worldwide. Previous studies suggested that metadherin (MTDH) is involved in the development of colorectal and lung ...cancers. However, how MTDH regulates the pathogenesis of these cancers remains largely unknown. Using genetically modified mouse models of spontaneous colorectal and lung cancers, we found that MTDH promotes cancer progression by facilitating Wnt activation and by inducing cytotoxic T-cell exhaustion, respectively. Moreover, we developed locked nucleic acid-modified (LNA) MTDH antisense oligonucleotides (ASO) that effectively and specifically suppress MTDH expression
and
. Treatments with MTDH ASOs in mouse models significantly attenuated progression and metastasis of colorectal, lung, and breast cancers. Our study opens a new avenue for developing therapies against colorectal and lung cancers by targeting MTDH using LNA-modified ASO. SIGNIFICANCE: This study provides new insights into the mechanism of MTDH in promoting colorectal and lung cancers, as well as genetic and pharmacologic evidence supporting the development of MTDH-targeting therapeutics.
Understanding the intrinsic mediators that render CD8
T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that ...C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8
T cells. Chop expression is increased in tumor-infiltrating CD8
T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8
T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8
T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8
T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity.
The adenosine axis contributes to the suppression of antitumor immune responses. The ectonucleotidase CD39 degrades extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which ...is degraded to adenosine by CD73. Adenosine binds to, e.g., the A2a receptor (A2aR), which reportedly suppresses effector immune cells. We investigated effects of ATP, AMP, and adenosine analogs on T cell proliferation, apoptosis, and proinflammatory cytokine secretion. CD39 and CD73 expression were suppressed using antisense oligonucleotides (ASOs), and A2aR was blocked using small molecules. Addition of ATP to T cells reduced proliferation and induced apoptosis. Intriguingly, those effects were reverted by suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogs did not suppress proliferation but inhibited secretion of proinflammatory cytokines. Here, we suggest that suppression of T cell proliferation is not directly mediated by A2aR but by intracellular downstream metabolites of adenosine, as blockade of the equilibrative nucleoside transporter (ENT) or adenosine kinase rescued proliferation and prevented induction of apoptosis. In conclusion, adenosine might primarily affect cytokine secretion directly via adenosine receptors, whereas adenosine metabolites might impair T cell proliferation and induce apoptosis. Therefore, inhibition of CD39 and/or CD73 has evident advantages over A2aR blockade to fully revert suppression of antitumor immune responses by the adenosine axis.
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The unleashing of antitumor immune responses by targeting the purine metabolism has emerged as a promising strategy in cancer immunotherapy. Festag and colleagues targeted different components of the purine metabolism using antisense oligonucleotides and small molecules and could show that T cell proliferation and cytokine release are differentially suppressed by distinct purine metabolites.
Cancer cells are known to develop mechanisms to circumvent effective anti-tumor immunity. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert ...extracellular immune-stimulating ATP to adenosine. CD39 is expressed by different immune cell populations as well as cancer cells of different tumor types and supports the tumor in escaping immune recognition and destruction. Thus, increasing extracellular ATP and simultaneously reducing adenosine concentrations in the tumor can lead to effective anti-tumor immunity.
We designed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) with specificity for human or mouse CD39 that do not need a transfection reagent or delivery system for efficient target knockdown. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and in primary human T cells. The effect of CD39 knockdown on ATP-degrading activity was evaluated by measuring levels of ATP in tumor cell supernatants and analysis of T cell proliferation in the presence of extracellular ATP. The in vivo effects of CD39-specific ASOs on target expression, anti-tumor immune responses and on tumor growth were analyzed in syngeneic mouse tumor models using multi-color flow cytometry.
CD39-specific ASOs suppressed expression of CD39 mRNA and protein in different murine and human cancer cell lines and in primary human T cells. Degradation of extracellular ATP was strongly reduced by CD39-specific ASOs. Strikingly, CD39 knockdown by ASOs was associated with improved CD8
T cell proliferation. Treatment of tumor-bearing mice with CD39-specific ASOs led to dose-dependent reduction of CD39-protein expression in regulatory T cells (Tregs) and tumor-associated macrophages. Moreover, frequency of intratumoral Tregs was substantially reduced in CD39 ASO-treated mice. As a consequence, the ratio of CD8
T cells to Tregs in tumors was improved, while PD-1 expression was induced in CD39 ASO-treated intratumoral CD8
T cells. Consequently, CD39 ASO treatment demonstrated potent reduction in tumor growth in combination with anti-PD-1 treatment.
Targeting of CD39 by ASOs represents a promising state-of-the art therapeutic approach to improve immune responses against tumors.
BackgroundImmune checkpoint inhibitors (ICI) are considered as a breakthrough in cancer therapy, however only a subset of patients actually responds to current treatments, indicating a strong ...remaining medical need. Locked nucleic acid (LNA) modified antisense oligonucleotides (ASOs) allow for a specific target knockdown after systemic injection without delivery agents. The membrane bound multi domain protein neuropilin-1 (NRP1) is a promising therapeutic target, since it has been implicated in various pro-tumorigenic processes, including stability of regulatory T-cells, exhaustion of CD8+ T-cells, macrophage migration and polarization, and neovascularization. Since these effects are mediated by different domains, blocking antibodies that only bind to a single domain might only have limited effects. Thus, the knockdown of the complete protein is conceptually advantageous.MethodsUsing our in-house Oligofyer™ bioinformatics system, we designed LNA-modified NRP1 specific ASOs and were able to identify potent candidates in cellular screens. The most promising candidates were tested by intraperitoneal injection in two syngeneic tumor models, either as a monotherapy, or in combination with an anti-PD-L1 antibody.ResultsIn syngeneic mouse models, systemically administered NRP1-specific ASOs achieved a potent target knockdown in several relevant cell types within the tumor microenvironment and a strong reduction in plasma levels of soluble NRP1. Furthermore we observed strongly reduced tumor growth-rates in animals treated with NRP1 ASOs as monotherapy. In combination with PD-L1 antibody we achieved a potent on-top effect with lasting complete tumor eradication in many animals and prolonged survival (figure 1). Re-challenge experiments with mice that were tumor-free after the treatment indicated establishment of anti-tumor immunity in treated mice.ConclusionsNRP1 is a promising new target with multiple pro-tumorigenic roles mediated by different domains. Simultaneous inhibition of all of these functions via knockdown of its expression by ASOs appears to be a favorable treatment modality in this regard, which could overcome limitations faced by alternative approaches. Systemically administered LNA-modified ASOs targeting NRP1 have the potential to become a new treatment option - both as a monotherapy and in combination with other ICIs - in various cancer indications that do not respond to current treatments. Further mechanistic research will be instrumental to translate these findings to the clinics.Abstract 881 Figure 1Effects on survival in orthotopic syngeneic EMT6 model
BackgroundAlthough immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of ...long-term efficacy may be due to a poor pre-existing network linking innate and adaptive immunity. Here, we present an antisense oligonucleotide (ASO)-based strategy that dually targets toll-like receptor 9 (TLR9) and programmed cell death ligand 1 (PD-L1), aiming to overcome resistance to anti-PD-L1 monoclonal therapy.MethodsWe designed a high-affinity immunomodulatory IM-TLR9:PD-L1-ASO antisense oligonucleotide (hereafter, IM-T9P1-ASO) targeting mouse PD-L1 messenger RNA and activating TLR9. Then, we performed in vitro and in vivo studies to validate the IM-T9P1-ASO activity, efficacy, and biological effects in tumors and draining lymph nodes. We also performed intravital imaging to study IM-T9P1-ASO pharmacokinetics in the tumor.ResultsIM-T9P1-ASO therapy, unlike PD-L1 antibody therapy, results in durable antitumor responses in multiple mouse cancer models. Mechanistically, IM-T9P1-ASO activates a state of tumor-associated dendritic cells (DCs), referred to here as DC3s, which have potent antitumor potential but express the PD-L1 checkpoint. IM-T9P1-ASO has two roles: it triggers the expansion of DC3s by engaging with TLR9 and downregulates PD-L1, thereby unleashing the antitumor functions of DC3s. This dual action leads to tumor rejection by T cells. The antitumor efficacy of IM-T9P1-ASO depends on the antitumor cytokine interleukin-12 (IL-12), produced by DC3s, and Batf3, a transcription factor required for DC development.ConclusionsBy simultaneously targeting TLR9 and PD-L1, IM-T9P1-ASO amplifies antitumor responses via DC activation, leading to sustained therapeutic efficacy in mice. By highlighting differences and similarities between mouse and human DCs, this study could serve to develop similar therapeutic strategies for patients with cancer.
Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against ...Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC50) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection.
Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the ...identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular
, and
experiments.
experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as
activity. Biochemical and
analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and
analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.
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