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Antisense oligonucleotides (ASOs) are a novel therapeutic strategy that targets a specific gene and suppresses its expression. The cryopyrin-associated periodic syndromes (CAPS) are a spectrum of autoinflammatory diseases characterized by systemic and tissue inflammation that is caused by heterozygous gain-of-function mutations in the nucleotide-binding and oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) gene. The aim of this study was to investigate the efficacy of an Nlrp3-specific ASO treatment in CAPS. An Nlrp3-specific ASO was designed and tested in murine cell lines and bone marrow–derived macrophages (BMDMs) from wild-type and CAPS mouse models. Nlrp3 knock-in mice were treated in vivo with Nlrp3-specific ASO, survival was monitored, and expression of organ-specific Nlrp3 and IL-1β was measured. Nlrp3-specific ASO treatment of murine cell lines and BMDMs showed a significant downregulation of Nlrp3 and mature IL-1β protein expression. Ex vivo treatment of Nlrp3 mutant mouse-derived BMDMs with Nlrp3-specific ASO demonstrated significantly reduced IL-1β release. In vivo, Nlrp3-specific ASO treatment of Nlrp3 mutant mice prolonged survival, reduced systemic inflammation, and decreased tissue-specific expression of Nlrp3 and mature IL-1β protein. The results of this study demonstrate that Nlrp3-specific ASO treatment downregulates Nlrp3 expression and IL-1β release in CAPS models, suggesting ASO therapy as a potential treatment of CAPS and other NLRP3-mediated diseases.

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Supplementary Material and Methods

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 in vitro and in vivo. 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.

Supplementary Material and Figure Legends

Sensitive in vivo imaging technologies applicable to the clinical setting are still lacking for adoptive T-cell–based immunotherapies, an important gap to fill if mechanisms of tumor rejection or escape are to be understood. Here, we propose a highly sensitive imaging technology to track human TCR-transgenic T cells in vivo by directly targeting the murinized constant TCR beta domain (TCRmu) with a zirconium-89 (89Zr)-labeled anti–TCRmu-F(ab')2 fragment. Binding of the labeled or unlabeled F(ab')2 fragment did not impair functionality of transgenic T cells in vitro and in vivo. Using a murine xenograft model of human myeloid sarcoma, we monitored by Immuno-PET imaging human central memory T cells (TCM), which were transgenic for a myeloid peroxidase (MPO)–specific TCR. Diverse T-cell distribution patterns were detected by PET/CT imaging, depending on the tumor size and rejection phase. Results were confirmed by IHC and semiquantitative evaluation of T-cell infiltration within the tumor corresponding to the PET/CT images. Overall, these findings offer a preclinical proof of concept for an imaging approach that is readily tractable for clinical translation. Cancer Res; 76(14); 4113–23. ©2016 AACR.

Investigation of different imaging time points to track TCR-transduced TCM after injection of 89Zr-aTCRmu-F(ab')2

Locked nucleic acid-modified antisense oligonucleotides (ASOs) can achieve strongly different degrees of target knockdown despite having similar biophysical properties and 100% homology with their target. The determinants for this observation remain largely unknown. We used multi-specific ASOs that have 100% sequence complementarity with a common target (IDO1) and a different number of diverse targets and investigated their effect on gene expression in a cell line by RNA-sequencing. We observed a significant higher chance for downregulation of long genes compared to short genes, of genes with high compared to lower expression, and of genes that have more than one binding site for the respective ASO. By investigating the expression of genes that have binding sites for more than one ASO we identified the individual binding site being an important determinant for activity. Under the selected experimental conditions we have not seen indications that availability of RNase H is a limiting factor as the number of degraded target RNA molecules correlated significantly with the number of predicted target RNA molecules. Taken together, by using multi-specific ASOs as tool compounds we identified determinants for ASO activity that can be taken into consideration to improve the selection process of highly potent and selective ASOs in the future.

Background: Maladaptive ER stress signaling in diabetic kidney disease (DKD) is linked to increased glomerular and tubular expression of the cell death-promoting transcription factor C/EBP homologous protein (CHOP). We determined whether therapy with locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) targeting CHOP ameliorates experimental DKD. Methods: Following an in vivo dose-escalation study, we determined the efficacy of CHOPASO in the early and later stages of experimental DKD (8- or 16-week-old db/db mice, respectively) alone or in combination with an angiotensin-converting enzyme inhibitor (ACEi). Renal functional parameters and morphological analyses were used to determine the effects. Renal gene expression profiling was conducted to determine differentially regulated genes and pathways. Several human CHOP-ASOs were tested in hyperglycemia-exposed human kidney cells. Results: CHOP-ASOs efficiently reduced renal CHOP expression in diabetic mice and reduced markers of DKD at early and late stages. Early combined intervention (CHOP-ASO and ACEi) efficiently prevented interstitial damage. At the later timepoint, the combined treatment reduced indices of both glomerular and tubular damage more efficiently than either intervention alone. A significantly larger number of genes and disease pathways were affected by CHOP-ASO, including reduced Slc5a2 (sodium-glucose transport protein 2) and PROM1 (CD133). Human CHOP-ASOs efficiently reduced glucose-induced CHOP and prevented cell death of human kidney cells in vitro Conclusions: The ASO-based approach efficiently reduced renal CHOP expression in a diabetic mouse model, providing an additional benefit to an ACEi in particular at later timepoints. These studies demonstrate that ASO-based therapies efficiently reduce maladaptive CHOP expression and ameliorate experimental DKD.

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 performedin vitroandin vivostudies 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, andBatf3, 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.

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 in vitro and in vivo. 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.

The field of antisense oligonucleotide therapeutics is rapidly growing and in addition to small molecules and therapeutic antibodies, oligonucleotide-based gene expression modifiers have been developed as fully accepted therapeutics. Antisense oligonucleotides are designed to modify gene expression of their specific target genes. However, as their effect relies on Watson–Crick base pairing, they could also bind to other unintended complementary RNAs showing sufficient sequence homology, which in turn could lead to off-target effects. It is assumed that these off-target effects depend on the degree of complementarity between the antisense oligonucleotides and off-target sequences. Aim of this study was the investigation of the effects of antisense oligonucleotides on the expression of potential off-targets having a defined number of mismatches to the oligonucleotide sequence. We extend recent studies by investigating the off-target profile of two 17-mer antisense oligonucleotides in two distinct human cell lines by a whole-transcriptome study using RNA sequencing. The relatively high percentage of significantly downregulated off-target genes for which one mismatch is present corroborates the requirement for intense bioinformatic screens and stringent specificity criteria to design antisense oligonucleotides with only minimal sequence complementarity to any non-target sequence. Avoiding suppression of off-target genes by a thorough bioinformatics screen should strongly reduce the risk for toxicities caused by antisense oligonucleotide-mediated off-target RNA suppression and finally result in safer antisense oligonucleotide-based therapeutics.

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., Graphical Abstract, 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.

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. Keywords: Ebola virus, antisense oligonucleotide, NPC1, LNA

Immune checkpoint inhibitors have become an important tool for the treatment of cancer. However, the majority of patients does not respond to the currently employed antibody-based treatments, indicating a need for additional targets and new treatment modalities. The invention of 3rd generation chemistries like locked nucleic acids (LNAs) has allowed the development of highly specific and efficacious antisense oligonucleotides (ASOs) that allow target knockdown in vivo without the need for complex delivery formulations. The membrane-bound protein neuropilin-1 (NRP1) was initially identified as a factor involved in cell migration, -survival, and neoangiogenesis making it an attractive target for cancer therapy. It was subsequently shown that it has additional immune-mediated pro-tumorigenic roles. It can suppress anti-tumor immune activity via regulation of trafficking of tumor-associated macrophages, phenotypic stability of Tregs, and exhaustion of effector T cells. Considering that these effects are mediated by multiple domains of NRP1, there is a conceptual advantage to down-regulate the expression of the whole protein over the functional or steric blockade of individual domains. We used our in-house Oligofyer™ bioinformatics system to design both human NRP1 specific LNAplus™ ASOs, as well as murine surrogates, which both achieved target knock-down of more than 85% in vitro. Furthermore, the murine surrogate down regulated NRP1 in tumor mouse models. Systemic application of mouse-specific ASOs without additional carriers or adjuvants has led to a strong delay of tumor growth or complete eradication in syngeneic mouse models. This effect was almost completely abrogated in immune-compromised NSG mice. Furthermore, re-challenge of immune-competent mice after tumor eradication did not result in new tumor growth, suggesting induction of a long-lasting immunity by LNAplus™ ASO-mediated knockdown of NRP1. Taken together, these encouraging results indicate that the ASO-mediated down-regulation of NRP1 has the potential to become a promising treatment option for patients in need. We are currently performing further experiments to fully elucidate the mechanisms that underlie the observed anti-tumor efficacy. Citation Format: Andre Maaske, Nicole Kirchhammer, Julia Festag, Laura Fernandez Rodriguez, Mélanie Buchi, Monika Schell, Stefanie Raith, Sven Michel, Richard Klar, Alfred Zippelius, Frank Jaschinski. Knock-down of Neuropilin-1 by locked nucleic acid antisense oligonucleotides facilitates cancer immune control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4149.

Background The so-called adenosine axis has emerged as a promising therapeutic target pathway as high adenosine levels in the tumor microenvironment contribute to the suppression of antitumor immune responses. The ectonucleotidases CD39 and CD73 act in concert to degrade extracellular immune-stimulating adenosine triphosphate (ATP) to immunosuppressive adenosine. According to the current model, subsequent suppression of effector immune cell function is caused by binding of adenosine to adenosine receptors like the A2a receptor (A2aR). The ectonucleotidases CD39 and CD73 as well as the A2aR have emerged as molecular targets within the adenosine axis with currently more than 20 clinical trials investigating antitumor effects of CD39-, CD73- or A2aR blockade. We aimed to perform a direct comparison of these targets with regard to their roles in regulating T-cell proliferation and IFN-γ secretion. Materials and Methods CD39 and CD73 expression was suppressed using LNAplusTM antisense oligonucleotides (ASOs). ASOs were synthesized as gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA, leaving a central gap for recruitment of the RNA-degrading enzyme RNaseH I. Knockdown efficacy of ASOs on mRNA and protein level was investigated in primary human T cells. Furthermore, the effects of ATP, AMP and adenosine analogues on T–cell proliferation and IFN–γ secretion were investigated. A2aR was blocked using small molecule inhibitors that are currently under clinical investigation. Results Treatment of human T cells with LNA-modified ASOs specific for human CD39 and CD73 resulted in potent target knockdown in vitro without the use of a transfection reagent. T-cell proliferation was reduced after addition of ATP to activated T cells that was completely reverted by ASO-mediated suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogues inhibited IFN–γ secretion of activated T cells, however, they did not suppress T-cell proliferation. Blockade of the adenosine kinase was able to revert the anti-proliferative effect of ATP degradation products, arguing for downstream metabolites of adenosine, but not A2aR signaling, being responsible for the suppression of T-cell proliferation. Conclusions Cytokine secretion and proliferation of T cells might be differentially regulated by the adenosine axis. Adenosine might primarily affect cytokine secretion via A2aR signaling, whereas adenosine metabolites might especially impair proliferation of activated T cells independent from A2aR signaling. Therefore, inhibition of CD39 and/or CD73 holds exceptional advantages over A2aR blockade as both, A2aR dependent and A2aR independent effects of ATP degradation products are targeted simultaneously. Disclosure Information J. Festag: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. T. Thelemann: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. M. Schell: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. S. Raith: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. S. Michel: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. R. Klar: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. F. Jaschinski: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG.

A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89Zr-Df-aTCRmu-F(ab')2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo. Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo. Local application of 89Zr-Df-aTCRmu-F(ab')2-labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x104 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89Zr-Df-aTCRmu-F(ab')2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x104 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.

Tumors can utilize a diverse repertoire of immunosuppressive mechanisms to evade attack by the immune system. Despite promising success with blockade of immune checkpoints like PD-1 the majority of patients does not respond to current immunotherapies. The degradation of tryptophan into immunosuppressive kynurenine is an important immunosuppressive pathway. Recent attempts to target the key enzymes of this pathway-IDO1 and TDO2-have so far failed to show therapeutic benefit in the clinic, potentially caused by insufficient target engagement. We, therefore, sought to add an alternative, highly efficient approach to block the degradation of tryptophan by inhibiting the expression of IDO1 and TDO2 using locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs). We show that LNA-modified ASOs can profoundly inhibit the expression of IDO1 and TDO2 in cancer cells in vitro without using a transfection reagent with IC50 values in the sub-micromolar range. We furthermore measured kynurenine production by ASO-treated cancer cells in vitro and observed potently reduced kynurenine levels. Accordingly, inhibiting IDO1 expression in cancer cells in an in vitro system leads to increased proliferation of activated T cells in coculture. We furthermore show that combined treatment of cancer cells in vitro with IDO1-specific ASOs and small molecule inhibitors can reduce the production of kynurenine by cancer cells in a synergistic manner. In conclusion, we propose that a combination of LNA-modified ASOs and small molecule inhibitors should be considered as a strategy for efficient blockade of the degradation of tryptophan into kynurenine in cancer immunotherapy.

Background 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. Methods 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. Results 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. Conclusion Targeting of CD39 by ASOs represents a promising state-of-the art therapeutic approach to improve immune responses against tumors. Electronic supplementary material The online version of this article (10.1186/s40425-019-0545-9) contains supplementary material, which is available to authorized users.

T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCR), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity of TCR-transgenic T cells in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate exemplarily for MPO that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.

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The microenvironment generated by tumor cells and suppressive stromal cells creates unfavorable conditions for effector immune cells in order to escape anti-tumor immunity. Besides suppressive pathways like for example the CD39/CD73 axis, mechanisms including nutrient starvation, hypoxia, exposure to high levels of reactive oxygen species and acidosis contribute to the suppression of tumor-reactive immune cells. We have recently discovered that the ER-stress response, in particular the C/EBP homologous protein (Chop) plays an important role in the suppression of tumor-exposed T cells. Furthermore, it is upregulated in activated T cells in vitro and may hamper the efficacy of adoptive T cell therapies. As Chop is a transcription factor it falls into the category of “difficult to drug” and therefore represents an optimal target for antisense-oligonucleotides. In order to revert Chop-induced suppression of T cell activity, we designed locked nucleic acid (LNA) ASOs with specificity for mouse or human Chop using our Oligofyer™ bioinformatics system. Knockdown on mRNA level was investigated in cancer cell lines and in T cells in vitro and the most potent ASOs were selected for further experiments. Downstream effects of Chop knockdown were investigated by mRNA expression analysis and flow cytometry. We furthermore investigated the effect of ex vivo Chop knockdown on the efficacy of adoptively transferred Pmel-specific T cells (recognized target: gp10025-33 peptide) in a B16 melanoma model. Treatment of cells with selected Chop-specific ASO leads to potent knockdown of Chop in vitro. In accordance with observations made in T cells derived from Chop-knockout mice, we observed an increase in T cell-associated transcription factor (Tbet), Interferon-gamma (IFN-γ) and Granzyme B (GZMB) expression in T cells that have been treated with a Chop ASO compared to control oligo treated cells. Strikingly, this translated into improved tumor control by Pmel-specific T cells. We furthermore observed an increase in IFN-γ producing tumor-infiltrating T cells and increased frequency of IFN-γ producing T cells in gp100 restimulated splenocytes from animals that received Chop ASO-treated Pmel-specific T cells. Of note, the impact of Chop knockdown was comparable to the effects that we observed in T cells derived from Chop knockout animals. Taken together, we herein show that Chop is a highly promising novel target in immunotherapy and can effectively be targeted by LNAplus™ ASOs. As shown in a model of adoptive T cell therapy, Chop ASOs have a high potential to optimize the efficacy of T cell therapies, such as chimeric antigen receptor (CAR)- or T cell receptor (TCR)-transgenic T cells. We are currently investigating the effect of systemic Chop ASO treatment in murine tumor models to expand the spectrum of applications of this innovative therapeutic tool. Citation Format: Richard Klar, Yu Cao, Eslam Mohamed, Sven Michel, Monika Schell, Lisa Hinterwimmer, Stefanie Raith, Paulo Rodriguez, Frank Jaschinski. Inhibition of ER-stress factor C/EBP homologous protein (Chop) with LNAplus™ antisense-oligonucleotides to improve immunotherapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3275.

The ectonucleotidases CD39 and CD73 have emerged as promising therapeutic targets to enhance antitumor immune responses, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on cancer cells and support the tumor in escaping immune recognition and destruction. Accordingly, in order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to reduce adenosine concentrations in the tumor microenvironment. As an alternative approach to small molecule inhibitors or antibodies, we developed antisense oligonucleotides (ASOs) using the OligofyerTM tool to suppress the expression of CD39 and CD73. ASOs were synthesized as Gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP. The impact of ASO-mediated target knockdown in vivo on anti-tumor immune responses was analyzed in a syngeneic mouse tumor model. Unformulated CD39- and CD73-specific ASOs achieved potent target knockdown on mRNA and protein level in tumor cell lines and primary human T cells in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability if T cells expressed CD39 or CD73. Notably, ASO-mediated knockdown of CD39 or CD73 reversed this inhibitory effect of ATP. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Treatment of tumor-bearing mice with CD39-specific ASO led to dose dependent reduction of CD39 protein expression e.g. in Tregs, tumor-associated macrophages and myeloid-derived suppressor cells. Strikingly, the frequency of intratumoral Tregs was considerably diminished in CD39 ASO-treated animals. Moreover, tumor growth was strongly reduced by CD39-specific ASO. Anti-tumor efficacy was improved when combining CD39 ASO with anti-PD-1 treatment. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors. Citation Format: Julia Festag, Tamara Thelemann, Abhishek S. Kashyap, Richard Klar, Sandra Kallert, Melanie Buchi, Lisa Hinterwimmer, Monika Schell, Stefanie Raith, Sven Michel, Alfred Zippelius, Frank Jaschinski. LNAplusTM antisense oligonucleotides targeting CD39 and CD73 have potent anti-tumor activity by modulating immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5021.

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., Neoantigens determine anti-cancer immunoreactivity and are important functional targets for immunotherapy. Here, the authors use deep mass spectrometry to characterize neoepitopes from human melanoma tissue and show the presence of tumour-reactive T cells with specificity for selected neoantigens.

The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion. In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction. After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/−ɣc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells. CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic.

Adoptive transfer of T cells genetically modified with tumour-specific T-cell receptors (TCR) is a promising novel approach in the treatment of cancer. We have previously isolated an allorestricted MHC class I-restricted TCR with specificity for Formin-like protein 1 (FMNL1) with potent activity against chronic lymphocytic leukaemia cells. CD4(+) T cells have been described to be highly important for tumour elimination although TCR derived from CD4(+) T cells with anti-tumour reactivity have been only rarely described. In this study we aimed to isolate MHC class-II-restricted CD4(+) T cells and TCR with specificity for leukaemia antigens. We used professional antigen-presenting cells pulsed with the leukaemia-associated and tumour-associated antigen FMNL1 for stimulation of autologous T cells in vitro. We isolated two CD4(+) HLA-DR-restricted T-cell clones and T-cell-derived TCR with so far unknown specificity but high reactivity against lymphoma cells and native malignant cells derived from HLA-matched patients with diverse leukaemias. Moreover, characterization of the TCR after TCR gene transfer revealed that specific characteristics of isolated TCR as reactivity in response to Toll-like receptors were transferable on effector cells. Our results have a major impact on the development of novel immunotherapies. They demonstrate that TCR with potent HLA-DR-restricted anti-leukaemic reactivity against so far undefined self-restricted antigens can be isolated from the healthy autorestricted CD4(+) T-cell repertoire and these TCR are highly interesting candidate tools for novel immunotherapies.

During the last decades it became obvious that the immune system can be utilized to evoke effective antitumor responses. However, cancer cells develop mechanisms to circumvent this. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to immunosuppressive adenosine. CD39 and CD73 are expressed on different immune cells as well as on a range of cancer cells and the latter are recognized to co-opt both ectonucelotidases for circumventing antitumor immune responses. In order to enhance immunity against tumors it would be favorable to increase extracellular ATP- and to simultaneously reduce adenosine concentrations in the tumor microenvironment. We designed antisense oligonucleotides (ASOs) with specificity for human and mouse CD39 and CD73. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and primary human T cells. CD39 and CD73 activity was evaluated by measuring levels of ATP, AMP and adenosine in cell supernatants. As functional readout we analyzed T cell proliferation and viability in presence of extracellular ATP and adenosine. The impact of ASO-mediated target knockdown in vivo on antitumor immune responses was analyzed in a syngeneic mouse tumor model. CD39- and CD73-specific ASOs suppressed expression of CD39 and CD73 mRNA and protein in different cancer cell lines and in primary human T cells without the need of any transfection reagent in vitro. Furthermore, degradation of extracellular ATP or AMP was significantly blocked by CD39- or CD73-specific ASOs while formation of adenosine was suppressed. Supplementation of cell culture medium with ATP impaired proliferation and viability of CD39- expressing CD8+ T cells. Strikingly, CD39-knockdown by ASO reversed the inhibitory effects of ATP on cell proliferation and viability. Extracellular AMP strongly inhibited proliferation and viability of CD73-expressing CD4+ T cells. Notably, AMP-mediated inhibition of proliferation was considerably less pronounced in cells treated with CD73-specific ASO. Systemic treatment of mice with CD39- and CD73-specific ASOs resulted in a knockdown of CD39 and CD73 mRNA expression in spleen and liver. Moreover, CD39-ASO treatment of tumor-bearing mice significantly reduced CD39-protein expression on tumor-associated macrophages, as well as intratumoral CD8+ T cells and Tregs. Remarkably, frequency of intratumoral Tregs was potently reduced by ASO-mediated CD39 knockdown. Taken together, targeting of CD39 and CD73 by ASOs represents a very promising state-of-the art therapeutic approach to improve immunity against tumors. Citation Format: Tamara Hilmenyuk, Sandra Kallert, Richard Klar, Lisa Hinterwimmer, Monika Schell, Marina Van Ark, Alfred Zippelius, Frank Jaschinski. Inhibition of Cd39 and Cd73 by 3rd-generation antisense oligonucleotides to improve immunity against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5909.

Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8(+) T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1(130) peptide-driven stimulations with HLA-A*02:01(+) dendritic cells (DC), allo-restricted HLA-A*02:01(-) CD8(+) T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01(-) primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2(-/-)γc(-/-) mouse model. Initially generated transgenic T cells specifically recognized STEAP1(130)-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01(+) ES lines more effectively than HLA-A*02:01(-) ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1(130)-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01(+) ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors.

Sensitive in vivo imaging technologies applicable to the clinical setting are still lacking for adoptive T-cell–based immunotherapies, an important gap to fill if mechanisms of tumor rejection or escape are to be understood. Here, we propose a highly sensitive imaging technology to track human TCR-transgenic T cells in vivo by directly targeting the murinized constant TCR beta domain (TCRmu) with a zirconium-89 (89Zr)-labeled anti–TCRmu-F(ab')2 fragment. Binding of the labeled or unlabeled F(ab')2 fragment did not impair functionality of transgenic T cells in vitro and in vivo. Using a murine xenograft model of human myeloid sarcoma, we monitored by Immuno-PET imaging human central memory T cells (TCM), which were transgenic for a myeloid peroxidase (MPO)–specific TCR. Diverse T-cell distribution patterns were detected by PET/CT imaging, depending on the tumor size and rejection phase. Results were confirmed by IHC and semiquantitative evaluation of T-cell infiltration within the tumor corresponding to the PET/CT images. Overall, these findings offer a preclinical proof of concept for an imaging approach that is readily tractable for clinical translation. Cancer Res; 76(14); 4113–23. ©2016 AACR.

Background: Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology targetable tumor associated antigens (TAA) are identified and exploited for engineered T cell therapy. Here, the specific recognition and lytic potential of transgenic, allo-restricted CD8+ T cells directed against the ES-associated antigen STEAP1 was examined. Methods: Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells, allo-restricted HLA-A*02:01- CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, TCR α- and β-chains were identified. They were cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01- primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2-/-γc-/- mouse model. Results: Initially generated and transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific IFNγ release. They lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01- ES lines. In vivo tumor growth was inhibited more effective with transgenic STEAP1130-specific T cells than with unspecific T cells. Conclusion: Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1 expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1 expressing tumors. Citation Format: David Schirmer, Richard Klar, Oxana Schmidt, Dirk Wohlleber, Wolfgang Uckert, Uwe Thiel, Felix Bohne, Dirk H. Busch, Angela M. Krackhardt, Stefan Burdach, Günther H. Richter. Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3202.