Immunogenic Form Research Articles

Immunogenicity of various variants of Ebola and Marburg virus glycoprotein genes in recombinant adenoviral vectors

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

Neoadjuvant lutetium PSMA, the TIME and immune response in high-risk localized prostate cancer.

High-risk localized prostate cancer remains a lethal disease with high rates of recurrence, metastases and death, despite attempts at curative local treatment including surgery. Disease recurrence is thought to be a result of failure of local control and occult micrometastases. Neoadjuvant strategies before surgery have been effective in many cancers, but, to date, none has worked in this setting for prostate cancer. Prostate-specific membrane antigen (PSMA)-based theranostics is an exciting and rapidly evolving field in prostate cancer. The novel intravenous radionuclide therapy, [177Lu]Lu-PSMA-617 (lutetium PSMA) has been shown to be effective in treating men with metastatic castration-resistant prostate cancer, targeting cells expressing PSMA throughout the body. When given in a neoadjuvant setting, lutetium PSMA might also improve long-term oncological outcomes in men with high-risk localized disease. A component of radiotherapy is potentially an immunogenic form of cancer cell death. Lutetium PSMA could cause cancer cell death, resulting in release of tumour antigens and induction of a tumour-specific systemic immune response. This targeted radioligand treatment has the potential to treat local and systemic tumour sites by directly targeting cells that express PSMA, but might also act indirectly via this systemic immune response. In selected patients, lutetium PSMA could potentially be combined with systemic immunotherapies to augment the antitumour T cell response, and this might produce long-lasting immunity in prostate cancer.

Natural variation of immune epitopes reveals intrabacterial antagonism

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Immunogenic cell death in cancer: targeting necroptosis to induce antitumour immunity.

Most metastatic cancers remain incurable due to the emergence of apoptosis-resistant clones, fuelled by intratumour heterogeneity and tumour evolution. To improve treatment, therapies should not only kill cancer cells but also activate the immune system against the tumour to eliminate any residual cancer cells that survive treatment. While current cancer therapies rely heavily on apoptosis - a largely immunologically silent form of cell death - there is growing interest in harnessing immunogenic forms of cell death such as necroptosis. Unlike apoptosis, necroptosis generates second messengers that act on immune cells in the tumour microenvironment, alerting them of danger. This lytic form of cell death optimizes the provision of antigens and adjuvanticity for immune cells, potentially boosting anticancer treatment approaches by combining cellular suicide and immune response approaches. In this Review, we discuss the mechanisms of necroptosis and how it activates antigen-presenting cells, drives cross-priming of CD8+ T cells and induces antitumour immune responses. We also examine the opportunities and potential drawbacks of such strategies for exposing cancer cells to immunological attacks.

Epigenetic Priming By Hypomethylation Enhances the Immunogenic Potential of Tolinapant in T-Cell Lymphoma

Background:Durable single-agent responses with the IAP antagonist, tolinapant (ASTX660), have been reported in peripheral T-cell lymphoma (PTCL) and cutaneous TCL (CTCL) patients (NCT02503423, Michot et al., EHA 2022). We have found that tolinapant has the potential to drive immunogenic form of cell death (ICD), such as necroptosis, in preclinical models of TCL (Ferrari et al., Blood Adv. 2021). Here, we further explored this by modulating key necrosome components in TCL cell lines and analysing changes in cell death signalling. We hypothesised the epigenetic mechanisms that limit induction of ICD could be reversed by combining tolinapant and hypomethylating agents, such as decitabine. Our biomarker-led approach demonstrated that this novel combination could overcome inherent resistance to a form of cell death that can engage a strong immune response. Materials and Methods: Key necrosome components were modified in human and mouse TCL cell lines by CRISPR knockout and activation, and effects of tolinapant on their viability measured in vitro. Promoter methylation was assessed by pyrosequencing following bisulfite treatment (EpigenDx). Effects of tolinapant and decitabine, as single-agents and in combination, on cytokine/chemokine release were assessed using Meso Scale Discovery (MSD) or Luminex assay. ICD biomarkers in cells and tumor lysates were analysed by ELISAs, MSD assays, qPCR and Western blotting. In vivo activity of tolinapant and decitabine were tested in caspase-8 knockout EL4 cells in a syngeneic setting. PTCL patient plasma samples were analysed by Human ExplorerMAP™ v. 1.0 (Luminex xMAP® technology). Results: Loss of necrosome components or necrosome inactivation led to reduced sensitivity of TCL cells to tolinapant. We confirmed decitabine treatment resulted in re-expression of necrosome proteins (e.g., RIPK3) and cancer testis antigens. Combination of decitabine and tolinapant led to increased lytic cell death over monotherapies, and this was accompanied by increased levels of interferon signalling and ICD biomarkers (e.g., HMGB1). Tolinapant treatment alone, or in combination with decitabine, led to elevated chemokine release from dying TCL cells. These observations indicate activation of necroptosis. In vivo, the combination treatment prolonged survival of mice bearing caspase-8 knockout EL4 tumors while monotherapies had limited effects. We also report that IP-10 (CXCL10) levels were increased in plasma samples of tolinapant-treated PTCL patients, as well as other plasma proteins, implying immune activation. Conclusions: We demonstrate that tolinapant acts as an immune modulator via promoting ICD, and this can be enhanced by epigenetic reprogramming with decitabine. These findings provide a rationale to support the on-going combination study of tolinapant with ASTX727 (oral decitabine and cedazuridine) that recently started in PTCL (ASCERTAIN-P, NCT05403450). It has also identified putative biomarkers of immunomodulatory activity that could be utilized in clinic.

Activation of autophagy by in situ Zn2+ chelation reaction for enhanced tumor chemoimmunotherapy.

Chemotherapy can induce a robust T cell antitumor immune response by triggering immunogenic cell death (ICD), a process in which tumor cells convert from nonimmunogenic to immunogenic forms. However, the antitumor immune response of ICD remains limited due to the low immunogenicity of tumor cells and the immunosuppressive tumor microenvironment. Although autophagy is involved in activating tumor immunity, the synergistic role of autophagy in ICD remains elusive and challenging. Herein, we report an autophagy amplification strategy using an ion-chelation reaction to augment chemoimmunotherapy in cancer treatments based on zinc ion (Zn2+)-doped, disulfiram (DSF)-loaded mesoporous silica nanoparticles (DSF@Zn-DMSNs). Upon pH-sensitive biodegradation of DSF@Zn-DMSNs, Zn2+ and DSF are coreleased in the mildly acidic tumor microenvironment, leading to the formation of toxic Zn2+ chelate through an in situ chelation reaction. Consequently, this chelate not only significantly stimulates cellular apoptosis and generates damage-associated molecular patterns (DAMPs) but also activates autophagy, which mediates the amplified release of DAMPs to enhance ICD. In vivo results demonstrated that DSF@Zn-DMSNs exhibit strong therapeutic efficacy via in situ ion chelation and possess the ability to activate autophagy, thus enhancing immunotherapy by promoting the infiltration of T cells. This study provides a smart in situ chelation strategy with tumor microenvironment-responsive autophagy amplification to achieve high tumor chemoimmunotherapy efficacy and biosafety.

Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer

BackgroundIncreasing evidence suggests that immunotherapy, especially immune checkpoint inhibitors (ICIs), has the potential to facilitate long-term survival in various cancer besides prostate cancer. Emerging evidence indicated that pyroptosis, an immunogenic form of cell death, could trigger an anti-tumor immune microenvironment and enhance the effectiveness of immunotherapy. Nevertheless, the mechanism underlying the regulation of pyroptosis signaling in prostate cancer remains unclear.MethodsThe differential expression of human E3 ligases in prostate cancer was integratedly analyzed from five independent public datasets. Moreover, the immunohistochemistry analysis of a tissue microarray derived from prostate cancer patients confirmed the results from the bioinformatic analysis. Furthermore, prostate cancer cell lines were evaluated via the next-generation RNA sequencing to assess transcriptomic profile upon CDC20 depletion. Next, qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay were employed to explore the correlation and interaction between CDC20 and GSDME. Both immune-deficient and immune-competent murine models were utilized to examine the anti-tumor efficacy of CDC20 inhibition with or without the anti-PD1 antibodies, respectively. To analyze the immune microenvironment of the xenografts, the tumor tissues were examined by immunohistochemistry and flow cytometry.ResultsThe analysis of multiple prostate cancer cohorts suggested that CDC20 was the most significantly over-expressed E3 ligase. In addition, CDC20 exerted a negative regulatory effect on the pyroptosis pathway by targeting GSDME for ubiquitination-mediated proteolysis in a degron-dependent manner. Knockdown of CDC20 leads to increased GSDME abundance and a transition from apoptosis to pyroptosis in response to death signals. Furthermore, in our syngeneic murine models, we found that depletion of CDC20 significantly enhances the anti-tumor immunity by promoting the infiltration of CD8+ T lymphocytes dependent on the existence of GSDME, as well as reducing myeloid immune cells. More importantly, Apcin, a small molecular inhibitor that targets CDC20, exhibited synergistic effects with anti-PD1-based immunotherapy in murine models of prostate cancer.ConclusionsOverall, these findings provide new insights into the upstream regulation of GSDME-mediated pyroptosis by CDC20, which specifically interacts with GSDME and facilitates its ubiquitination in a degron-dependent manner. Importantly, our data highlight novel molecular pathways for targeting cellular pyroptosis and enhancing the effectiveness of anti-PD1-based immunotherapy.

#4967 GASDERMIN D IS REQUIRED FOR CONTROL OF NECROPTOTIC CELL DEATH IN AKI

Abstract Background and Aims Contradicting previous reasoning, it is now established that necrotic rather than apoptotic cell death pathophysiologically drives acute tubular necrosis during AKI. Whereas involvement of necroptosis and ferroptosis has been demonstrated by various groups, the role of pyroptosis, a highly immunogenic form of cell death requiring proteolytic activation of gasdermin protein family members, remains uncertain upon AKI. Thus, we aimed to investigate the role of pyroptosis and its mechanism of action in AKI. Method We set to detect gasdermin D (GSDMD) expression in murine renal samples after ischemia/reperfusion injury (AKI) by immunohistochemistry. Furthermore, we investigated GSDMD-deficient mice in IRI and cisplatin-induced AKI. To conclude on mechanisms, we isolated fresh murine renal tubules and measured LDH release over time. We generated MLKL/GSDMDdko mice to investigate the interplay of these caspase-dependent forms of regulated necrosis in AKI. Results After IRI, we detected a specific GSDMD signal in areas surrounding necrotic tubules, whereas no such signal could be detected within necrotic areas. Whereas interference with other forms of regulated necrosis leads to reduced injury, GSDMD-deficient mice demonstrated higher levels of serum creatinine and urea as well as more severe tubular injury compared to wildtypes. In line with the findings of immunohistochemistry, we only found GSDMD protein expression in whole kidney lysates but not isolated renal tubules; and no difference in spontaneous cell death propagation was detectable. Co-deletion of MLKL (effector protein of necroptosis) reversed the sensitization to AKI by GSDMD-deficiency. These results could be reproduced in cisplatin-induced AKI. Conclusion Here, we found an unexpected protective role for GSDMD, the effector protein of pyroptosis, in two different models of AKI. Mechanistically, our studies indicate the effect of GSDMD to function outside the tubular compartment, specifically surrounding areas of tubular necrosis. These infiltrating innate immune cells appear to interfere with tubular necroptosis in a non-cell autonomous manner. Alongside with these mechanistic insights, our data urge caution when inhibition of pyroptosis is therapeutically considered.

Brincidofovir induces potent anti‐tumor activity in MYC‐driven Natural Killer/T‐cell Lymphoma with loss of transcriptional repressor TLE1

Introduction: Natural killer/T-cell lymphoma (NKTCL) is an aggressive Epstein-Barr virus (EBV)-associated malignancy which is prevalent in Asian countries. Brincidofovir (BCV), a lipid conjugated form of cidofovir, is a biologically-active acyclic nucleoside phosphonate recently shown to exhibit both antiviral and antitumor properties. Methods: We investigated BCV in NKTCL cell-lines (n = 11) and mouse xenograft models. Whole exome sequencing, bulk RNAseq and single cell RNAseq (scRNAseq) were employed to examine mechanisms of action, and validated using orthogonal assays. Results: BCV inhibited viability in all NKTCL cell-lines in a dose- and time-dependent manner. Highest sensitivity was demonstrated in four cell-lines KAI-3, NK-S1, NK-92 and KHYG-1 (IC50 36.0 to 303.6 ng/ml). Intraperitoneal BCV (40 mg/kg, 2X per week) inhibited tumor growth in NOD/SCID mice NK-S1 xenografts, compared with vehicle alone (p = 0.0005, two-tailed t-test). Whole exome sequencing of the cell-lines revealed widespread mutations in the JAK/STAT and DNA repair pathways, with no clear correlation observed with BCV sensitivity. RNAseq and Hallmark gene set enrichment analysis showed that MYC target pathways (FDR q < 0.001) were prominently upregulated in the four sensitive cell-lines compared to the rest. RNAseq of BCV-treated cell-lines (KAI-3 and NK-S1) revealed striking downregulation of MYC target pathways. scRNAseq revealed distinct temporal cell states evoked by BCV, including appearance of cell clusters enriched for p53 and apoptosis pathways, cell cycle and DNA repair pathways, as well as type I/II interferon and cytokine signaling. Western blot and quantitative PCR showed that markers of replication stress, DNA damage and STING signaling were potently upregulated, implicating an immunogenic form of cell death. Notably, TLE1, a known transcriptional repressor of the MYC oncogene, was the topmost downregulated gene (log2FoldChange -7.39, adjusted p = 3.51E-31) in the four BCV-sensitive cell-lines on RNAseq. This finding was verified on qPCR (p = 0.0061, Mann-Whitney test) and Western blot. In keeping with this result, RNAseq on NKTCL patient samples (n = 36) showed that TLE1-low tumors were enriched for MYC target pathways, which conferred worse progression-free survival (HR 3.44, 95% CI: 1.23–10.49, p = 0.0301). Conclusions: Taken together, these results suggest that BCV may play a novel role in the treatment of MYC-driven NKTCL. Loss of TLE1, a putative predictive biomarker, implies that BCV may favour patients in this poor prognostic subgroup. The research was funded by: Symbio Pharmaceuticals, Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore Ministry of Health’s National Medical Research Council Transition Award (TA21jun-0005), Research Training Fellowship Seed Fund (SEEDFD21jun-0002), Large Collaborative Grant (OFLCG18May-0028), and Collaborative Centre Grant (TETRAD II). Keywords: Diagnostic and Prognostic Biomarkers, Extranodal non-Hodgkin lymphoma, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. J. Y. Chan Consultant or advisory role: Symbio Pharmaceuticals Research funding: Symbio Pharmaceuticals M. Hazama Employment or leadership position: SymBio Pharmaceuticals Limited K. Fukushima Employment or leadership position: SymBio Pharmaceuticals Limited C. K. Ong Research funding: SymBio Pharmaceuticals Limited

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl protein transferase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC), but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12, was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naïve T cells, and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells, engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Abstract 6144: Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis

Abstract Once cancer has metastasized it remains incurable owing to evolved resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by polyploidization of the aneuploid cancer genome. We have shown that this polyploidization is accomplished through endocycling that leads to an enlarged cell size with increased transcriptomic and protein content, higher oxidative buffer capacity, and increased autophagy. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to multiple stressors in vitro, and are resistant to subsequent treatment with several classes of chemotherapy. Thus, we hypothesize that the PACC state is driving therapeutic resistance to current treatment regimens. Elimination of this overlooked phenotype is critical for sustained anticancer therapy response. Cancer cells, including those in the PACC state, control canonical cell cycle and cell death programs to evade apoptosis from external stressors including chemotherapy, highlighting an opportunity to leverage cell cycle-independent death pathways as an alternative therapeutic strategy1. Ferroptosis is a cell cycle agnostic immunogenic form of cell death that is an ideal pathway for elimination of cells in the PACC state. Characterization of ROS levels, glutathione content, labile iron homeostasis, and lipid content indicate that cells in the PACC state have a mounting susceptibility to ferroptosis. Our data suggests that cells in the PACC state keep ferroptosis in check through elevated levels of reduced glutathione and proficient redox activity, including turn over of GPX4, the sole enzyme counteracting lipid peroxidation. Direct inhibition of GPX4 is highly toxic to cells in the PACC state, particularly at later timepoints post therapy removal when the immediate stress response is largely resolved. These studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state with the intention of eliminating cancer resistance. 1Loftus, L.V.; Amend, S.R.; Pienta, K.J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 4723. https://doi.org/10.3390/ijms23094723 Citation Format: Luke Loftus. Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6144.

Exosomes in COVID‐19 infection: Focus on role in diagnosis, pathogenesis, immunity, and clinical trials

Since the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread as a new strain of coronavirus disease (COVID-19) and progressed as a global pandemic. Exosomes are membrane-bound vesicles released from almost all cells and are crucially involved in cell-cell communication. Interestingly, COVID-19 viral particles produce exosomes that moderate communication between infected and uninfected cells. Hence, there is growing evidence highlighting the crucial implications of exosomes in COVID-19 infection, transmission, intercellular spread, and reinfection. On the other hand, clinical trials have demonstrated mesenchymal stem cell-derived exosomes as a promising therapeutic strategy for severely affected COVID-19 patients. Also, convalescent plasma-derived exosomes have been proposed for multiple efficacies in COVID-19 patients. Furthermore, messenger RNAs (mRNA)-loaded exosomes were superior to mRNA-loaded lipid nanoparticles as a delivery system. Hence, exosomes can be used to safely induce SARS-CoV-2 immunity via their loading with mRNAs encoding immunogenic forms of SARS-CoV-2 spike and nucleocapsid proteins. Moreover, exosomes can be used as a nano-delivery system for microRNA to alleviate cytokine storm and prevent the progression of organ failure in COVID-19 patients. The present review summarizes state of the art concerning the role of exosomes in COVID-19 infection and accompanying organ complications as well as the potential use of exosomes in COVID-19 diagnosis, treatment, drug delivery, and vaccination. The review also sheds the light on the common biogenic pathway between the SARS-CoV-2 virus and exosomes. Additionally, the latest and current clinical trials using exosomes for COVID-19 infection are summarized.

RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma.

Radiotherapy induces DNA damage, resulting in cell-cycle arrest and activation of cell-intrinsic death pathways. However, the radioresistance of some tumour entities such as malignant melanoma limits its clinical application. The innate immune sensing receptor retinoic acid-inducible gene I (RIG-I) is ubiquitously expressed and upon activation triggers an immunogenic form of cell death in a variety of tumour cell types including melanoma. To date, the potential of RIG-I ligands to overcome radioresistance of tumour cells has not been investigated. Here, we demonstrate that RIG-I activation enhanced the extent and immunogenicity of irradiation-induced tumour cell death in human and murine melanoma cells in vitro and improved survival in the murine B16 melanoma model invivo. Transcriptome analysis pointed to a central role for p53, which was confirmed using p53-/- B16 cells. In vivo, the additional effect of RIG-I in combination with irradiation on tumour growth was absent in mice carrying p53-/- B16 tumours, while the antitumoural response to RIG-I stimulation alone was maintained. Our results identify p53 as a pivotal checkpoint that is triggered by RIG-I resulting in enhanced irradiation-induced tumour cell death. Thus, the combined administration of RIG-I ligands and radiotherapy is a promising approach to treating radioresistant tumours with a functional p53 pathway, such as melanoma.

Alternative exon usage in TRIM21 determines the antigenicity of Ro52/TRIM21 in systemic lupus erythematosus.

The origin and mechanisms of autoantigen generation in systemic lupus erythematosus (SLE) are poorly understood. Here, we identified SLE neutrophils activated in vivo by IFN as a prominent source of Ro52, also known as tripartite motif-containing protein 21 (TRIM21), a critical autoantigen historically thought to be primarily generated by keratinocytes in SLE. Different from mononuclear cells and keratinocytes, SLE neutrophils are enriched in several unique Ro52 species containing a core sequence encoded by exon 4 (Ro52Ex4) in TRIM21. Ro52Ex4 is the main target of anti-Ro52 antibodies and is found in 2 Ro52 variants (Ro52α and a potentially novel isoform termed Ro52γ) upregulated in SLE neutrophils. Further analysis of Ro52γ revealed a subset of autoantibodies against a unique C-terminal domain (Ro52γCT) generated from a frameshift due to the lack of exon 6 in Ro52γ. Antibodies to Ro52Ex4 and Ro52γCT distinguish SLE patient subsets characterized by distinct clinical, laboratory, treatment, and transcriptional profiles that are not discerned by the "classical" anti-Ro52 antibodies. These studies uncover IFN-activated neutrophils as a key source of unique immunogenic forms of Ro52 in SLE. Moreover, the finding of Ro52Ex4 and Ro52γCT as core targets of anti-Ro52 antibodies focus interest on Ro52γ as the potential isoform toward which immunological tolerance is initially lost in SLE.

P1293: COMBINING THE IAP ANTAGONIST, TOLINAPANT, WITH A DNA HYPOMETHYLATING AGENT ENHANCES ANTI-TUMOUR MECHANISMS IN PRECLINICAL MODELS OF T-CELL LYMPHOMA.

Background: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In a Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in preclinical studies. Aims: Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death, such as necroptosis, by rational combination of our clinical compounds in preclinical models of TCL. Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key necroptosis biomarkers (RIPK3, MLKL) were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. Karpas-299 cells genetically-manipulated to express RIPK3 were used to demonstrate involvement of necroptosis in drug-induced cell death (Cytotox NIR) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key immunomodulatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma. Levels of methylation in RIPK3 promoter regions were measured by pyrosequencing after bisulfite conversion. Comparative changes in gene expression were measured by RT-qPCR. Results: TCL cell lines treated with tolinapant, decitabine or both led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating target engagement of both agents. The combination of tolinapant and decitabine synergistically reduced viability of some human T-cell lymphoma cell lines. Some cell lines, including Karpas-299, were resistant to tolinapant treatment and showed low expression of RIPK3, which was found to be due to promoter methylation. Increased expression of RIPK3 in Karpas-299 by genetic manipulation or by decitabine treatment resulted in enhanced lytic cell death upon tolinapant treatment. Decitabine and tolinapant treatments resulted in expression of cytokines, chemokines and DAMPs, suggesting potential for immune activation and the effects were enhanced when combined. Furthermore, normally silenced cancer/testis antigen expression was increased by decitabine, potentially increasing the immunogenicity of the cells. Evaluation of the combination of agents in mouse models suggested that increased necroptosis signal and immune-potentiating biomarker modulation can be achieved in vivo. Summary/Conclusion: These data demonstrate that hypomethylating agents enhance immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. In addition, modulation of cytokine response and cancer/testis antigen expression could enhance anti-tumour response. These findings provide a strong rationale to explore this combination clinically.

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection.

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4-14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.

DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

SummaryPathogens including autoantigens all failed to induce systemic lupus erythematosus (SLE). We, instead, studied the integrity of host's immune response that recognized pathogen. By stimulating TCR with an antigen repeatedly to levels that surpass host's steady-state response, self-organized criticality, SLE was induced in mice normally not prone to autoimmunity, wherein T follicular helper (Tfh) cells expressing the guanine nucleotide exchange factor DOCK8 on the cell surface were newly generated. DOCK8+Tfh cells passed through TCR re-revision and induced varieties of autoantibody and lupus lesions. They existed in splenic red pulp and peripheral blood of active lupus patients, which subsequently declined after therapy. Autoantibodies and disease were healed by anti-DOCK8 antibody in the mice including SLE-model (NZBxNZW) F1 mice. Thus, DOCK8+Tfh cells generated after repeated TCR stimulation by immunogenic form of pathogen, either exogenous or endogenous, in combination with HLA to levels that surpass system's self-organized criticality, cause SLE.

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. DisclosuresSims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

Antagonism of inhibitors of apoptosis proteins reveals a novel, immune response-based therapeutic approach for T-cell lymphoma

Tolinapant (ASTX660) is a potent, nonpeptidomimetic antagonist of cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) and X-linked IAP, which is currently being evaluated in a phase 2 study in T-cell lymphoma (TCL) patients. Tolinapant has demonstrated evidence of single-agent clinical activity in relapsed/refractory peripheral TCL and cutaneous TCL. To investigate the mechanism of action underlying the single-agent activity observed in the clinic, we have used a comprehensive translational approach integrating in vitro and in vivo models of TCL confirmed by data from human tumor biopsies. Here, we show that tolinapant acts as an efficacious immunomodulatory molecule capable of inducing complete tumor regression in a syngeneic model of TCL exclusively in the presence of an intact immune system. These findings were confirmed in samples from our ongoing clinical study showing that tolinapant treatment can induce changes in gene expression and cytokine profile consistent with immune modulation. Mechanistically, we show that tolinapant can activate both the adaptive and the innate arms of the immune system through the induction of immunogenic forms of cell death. In summary, we describe a novel role for IAP antagonists as immunomodulatory molecules capable of promoting a robust antitumor immune response in TCL.