Enhanced Antigen Presentation Research Articles

Interleukin-17A knockout or self-recovery alleviated autoimmune reaction induced by fluoride in mouse testis

Fluoride (F) is usually treated as a hazardous material, and F-caused public health problem has attracted global attention. Previous studies demonstrate that interleukin-17A (IL-17A) plays a crucial role in F-elicited autoimmune orchitis and self-recovery reverses F-induced testicular toxicity to some extent, but these basic mechanisms remain unclear. Thus, we established a 180 d F exposure model of wild type (WT) mice and IL-17A knockout mice (C57BL/6 J background), and 60 d & 120 d self-recovery model based on F exposure model of WT mice, and used various techniques like qRT-PCR, western blot, immunohistochemistry and ELISA to further explore the mechanism of F-induced autoimmune reaction, the role of IL-17A in it and the reversibility of F-caused toxicity in testis. The results indicated that F exposure for 180 d caused the decreased sperm quality, the damaged testis histopathology, the enhanced mRNA and protein expression levels of inflammatory cytokines, the changes of autoantibody such as the appearance and increased content of anti-testicular autoantibodies in sera and the autoantibody deposition in testis, the alterations of autoimmune related genes containing the decreased mRNA and protein expressions of AIRE and FOXP3 with an increase of MHCII, and the reduced protein expressions of CTLA4, and the activation of IL-17A signaling cascade like the elevated mRNA and protein expressions of IL-17A, Act1, NF-κB, AP-1 and CEBPβ, and the increased protein expressions of IL-17RC, with a decrease of IκBα. After IL-17A knockout, 29 of 35 F-induced changes were alleviated. In two self-recovery models, all F-caused differences except fluorine concentration in femur were gradually restored in a time-dependent manner. This study concluded that IL-17A knockout or self-recovery attenuated F-induced testicular injury and decrease of sperm quality through alleviating autoimmune reaction which was involved with the activation of IL-17A pathway, the damage of self-tolerance and the enhancement of antigen presentation.

Abstract LB279: Targeting microglial metabolic rewiring synergizes with immune checkpoint blockade therapy for glioblastoma

Abstract Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)-dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2 +/−) or microglia-specific Nr4a2 (Nr4a2 fl/fl Cx3cr1 cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacological NR4A2 inhibition attenuated the pro-tumorigenic TIME, and targeting the NR4A2 or SQLE enhanced therapeutic efficacy of immune checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2-SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer. Citation Format: Zengpanpan Ye, Xiaolin Ai, Linjie Zhao, Jeremy N. Rich, Shengtao Zhou. Targeting microglial metabolic rewiring synergizes with immune checkpoint blockade therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB279.

Abstract 6384: ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression

Abstract Background: Dendritic cells (DCs) are vital for initiating antigen-specific T cell-dependent antitumor immune responses. However, emerging evidence reveals that local DCs within the tumor microenvironment (TME) are tolerized to facilitate immune evasion. Reprogramming of DCs is recognized as a promising strategy for tumor immunotherapy. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor belonging to the LILR family which is mainly expressed on normal myeloid cells and myeloid-derived malignant cells. It is upregulated on tolerogenic DCs (tolDCs), which exhibit low expression levels of costimulatory molecules and resistance to DC maturation. Targeting LILRB4 to reprogram tolDCs has been reported to be a promising strategy for cancer treatment. Here we report the preclinical development of an LILRB4 antagonist antibody, ATG-034. Method: The protein-based and cell-based binding affinity of ATG-034 were measured using SPR, ELISA and FACS analysis. Fc receptor (FcR) stimulation assay was used to evaluate the ability of ATG-034 to block the interaction of LILRB4 with its ligand fibronectin. ATG-034-mediated enhancement of antigen presentation and co-stimulation ability of DCs was determined by FACS analysis for the change of surface expression of HLA-DR, HLA-ABC, CD86, and CD206. The mixed lymphocyte reaction (MLR) assay was used to assess the immunomodulatory potential of ATG-034. The in vivo antitumor efficacy of ATG-034 was evaluated in a radiation therapy-resistant murine Lewis lung carcinoma (LLC) syngeneic model. Results: ATG-034 binds to LILRB4 protein with a single-digit nM affinity. It potently reversed the fibronectin-mediated inhibition of FcR-driven TNF-α cytokine production. TolDCs were reprogrammed by ATG-034 to immunogenic DCs with significant upregulation of the expression of HLA-DR, HLA-ABC, CD86 and downregulation of the expression of CD206. Furthermore, ATG-034 remarkably reinvigorated tolDCs for priming T cell activation with noticeable upregulation of the expression of CD25 and the secretion of IFN-γ, enhancing antitumor immunity. In addition, 10 mg/kg ATG-034 significantly inhibited the LLC tumor growth in vivo with a TGI of 40.28%, while a benchmark antibody only demonstrated a 24.43% TGI. Conclusion: Our data show that ATG-034 reprograms tolDC, enhancing anti-tumor immunity, and demonstrates potent in vivo anti-tumor efficacy. Therefore, ATG-034 may be a promising strategy for the treatment of cancer. Citation Format: Ao Sun, Enlin Zheng, Huili Cao, Mengshi Cao, Suya Bai, Peng Chen, Linjie Tian, Jay Mei, Bo Shan, Bing Hou. ATG-034, an LILRB4 antagonist antibody, reinvigorates dendritic cells and prevents tumor progression [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 6384.

Abstract 6366: NB002, a novel therapeutic antibody targeting unique epitope on TIM-3 presenting potent antitumor activity

Abstract Antibody-based Immune checkpoint inhibitor (ICI) therapies targeting cytotoxic T lymphocyte antigen-4 (CTLA-4) or programmed death receptor-1 (PD-1) have significantly improved clinical outcomes in cancer treatment. Nonetheless, a large portion of the population has limited benefit from these ICI treatments due to lack of efficacy, resistance and intolerance. T-cell immunoglobulin and mucin domain containing-3 (TIM-3) has been proposed as a target for cancer immunotherapy. It is an inhibitory receptor modulating immune tolerance, and its expression has been broadly reported to be closely correlated to T cell exhaustion and pessimistic prognosis. Besides negatively modulating T cell status, TIM-3 is also expressed on natural killer (NK) cells, dendritic cells (DCs), macrophages and regulates immune responses bridging innate immunity and adaptive immunity. NB002 is a novel humanized Fc-engineered IgG1κ antibody, developed via NeologicsBio’s integrated target validation & antibody screening Tier-A platform. It has been characterized in a serial of function and efficacy studies and completed IND-enabling preclinical development. NB002 binds to human TIM3 with high affinity in the low nanomolar range and antagonize the TIM-3 pathway effectively in reporter cell and primary cell assays. NB002 increased the secretion of IFN-γ by activated PBMC cells and enhanced activation and killing activity of NK cells. Moreover, it exhibited a distinctive activity of restoring myeloid cells and enhancing antigen presentation and processing during anti-tumor immune responses, and superior efficacy in multiple humanized mouse models of cancer as monotherapy regimen. Importantly, X-ray crystallography demonstrated that NB002 recognized a unique epitope on TIM-3 and was confirmed by epitope binning assay and structural alignment. These data could potentially explain novel features of NB002, restoring both acquired and innate immunity which could lead cold tumor to hot tumor.In summary, these data showed that NB002 is a potent anti-TIM-3 antibody with pre-clinical properties. A phase 1 study is planned and patients with advanced metastatic solid tumors will be recruited. Citation Format: Yu Zhang, Jinyu Dong, Binbin Wang, Jie Ni, Dong Wang, Liegang Shao, Meiguang Xiong, Ting Wang, Florencia McAllister, Xin Dong. NB002, a novel therapeutic antibody targeting unique epitope on TIM-3 presenting potent antitumor activity. [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 6366.

Abstract 3089: XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies

Abstract Enhancer of zeste homolog 2 (EZH2) is the histone methyltransferase component of polycomb repressive complex 2 (PRC2) that can regulate gene expression by H3K27 trimethylation (H3K27Me3). EZH2 also mediates PRC2-dependent non-histone protein methylation or directly methylates non-histone proteins to regulate gene expression independent of PRC2. EZH2 plays an important role in regulation of cell cycle, autophagy, apoptosis and cell senescence. Mutated or overexpressed EZH2 associates with cancer initiation, progression, drug resistance and immunity regulation in cancer. XNW5004 is a rationally designed selective EZH2 inhibitor which potently inhibits both wild type and mutant EZH2. XNW5004 significantly suppresses level of H3K27Me3, arrests cell cycle in G1/S phase and promotes apoptosis in tumor. XNW5004 exhibits strong antitumor activity in EZH2-mutant lymphoma xenograft models, such as Karpas422 (Y641N), Pfeiffer (A677G) and SU-DHL-10 (Y641F). Since inhibition of EZH2 synthetically lethal in SWI/SNF subunits mutated cancers, XNW5004 exhibits dose-dependent efficacy in solid tumor models such as G401 (SMARCB1 deficient) and COV434 (SMARCA2/SMARCA4 deficient) cancer cell lines and xenografts. In prostate cancer cells, EZH2 can bind to the promoter of androgen receptor (AR) gene and activate AR expression. In PARPi-treated cancer cells, inhibition of PARP (poly-ADP-ribose polymerase) diminishes PARylation of EZH2 to result in reactivation of EZH2 and drug resistance to PARPi. In tumor microenvironment, inhibition of EZH2 promotes differentiation of T cells and NK cells, enhances antigen presentation ability of dendritic cells, and increases cytokine/chemokine secretion to recruit T cells into tumor tissue. Based on the above mechanisms, XNW5004 could potentially improve efficacy of ARi, PARPi and anti-PD-1. We have verified the synergistic effects of those combinations in multiple solid tumor xenograft models. In pharmacokinetic studies, XNW5004 has excellent oral bioavailability and exposure. XNW5004 shows good toxicological profile as well, has a wide therapeutic window in rats and dogs, and the risk of clinical safety was controllable. In summary, XNW5004 is a potent EZH2 inhibitor with promising efficacy, pharmacokinetic and safety properties. XNW5004 is expected to be a potent therapeutic candidate in cancer treatment and is now in Phase II clinical trial for both liquid and solid tumors. Citation Format: Yonghan Hu, Wei Peng, Zhe Zhang, Zhenwei Wu, Xi Chen, Yan Huang, Xin Li, Cungang Liu, Xiaojun Liu, Qifeng Shi, Xufang Wang, Wengui Wang, Haiyang Wei, Yuchuan Wu, Jun Xian, Linfeng Xu, Lina Zhang, Jinfeng Zhao, Meijie Le, Jing Qiang. XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies [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 3089.

Abstract 5119: Nano-immunotherapy targeting PD-L1, PLK1, and TLR9 for treatment of non-small cell lung cancer

Abstract Given the complexity of cancer, it is becoming apparent that combination therapy - in which complementary biological pathways are simultaneously targeted - is required to achieve clinically meaningful responses. To that end, PDX Pharma, in collaboration with Oregon Health & Science University, has developed a proprietary nanoparticle platform (Pdx-NP™) that can effectively co-deliver a plethora of therapeutic modalities while maintaining a small size in saline (100 nm), suitable for infusion and tumor accumulation. This enables the targeting of complementary cancer and immune pathways, leading to synergistic clinical benefits. Herein, we report on the second generation ARAC-02 (Antigen Release Agent and Checkpoint Inhibitor) which is designed to improve the efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). While ICIs have improved survival in a subset of patients with advanced NSCLC, only a minority of patients respond to ICIs, highlighting the need for combination immunotherapy. ARAC-02 co-delivers a polo-like kinase 1 (PLK1)-targeted therapy (volasertib), a PD-L1 antibody, and the immune-stimulant CpG. These three agents were carefully selected based on their roles in cancer and immune pathways. Volasertib 1) selectively kills cancer cells, 2) modulates the immune-suppressive tumor microenvironment, and 3) upregulates PD-L1 expression in cancer cells, providing opportunity for targeted delivery with PD-L1 antibody on the nanoparticles. CpG is a TLR9 agonist that enhances antigen presentation to generate tumor-specific T cells. PD-L1 antibodies serve as tumor targeting agent for nanoparticle delivery as well as an ICI (i.e., PD-L1 degradation upon internalization with the nanoparticles), releasing the brakes and allowing T cells to attack the cancer. We found that the optimized ARAC-02 is preferentially internalized in PD-L1 expressing NSCLC cells, demonstrating a 10-fold greater uptake to PD-L1(high) NSCLC cells than PD-L1(low) normal cells. ARAC-02 effectively kills NSCLC cells regardless of their mutational status, while sparing normal cells. In a metastatic lung tumor model, ARAC-02 treatments reduced the tumor burden by 30-fold vs. saline control and 8-fold vs. the first generation ARAC (not containing CpG), whose effect was mainly mediated by CD8+ T cells. ARAC-02 was found to be safe to mice as assessed by body weight, serum biomarkers, and histology. Importantly, intravenous infusions of the platform was also found to be safe in a preliminary toxicology study in non-human primates. Due to its unique ability to catalyze various steps of the adaptive immune response, ARAC-02 is anticipated to provide superior outcomes in NSCLC and a broad range of tumor types regardless of baseline PD-L1 expression. Citation Format: Moataz Reda, Worapol Ngamcherdtrakul, Ruijie Wang, Noah Crumrine, Cole Baker, Alyssa Wallstrum, Jeremy Saito, Natalie White, Wassana Yantasee. Nano-immunotherapy targeting PD-L1, PLK1, and TLR9 for treatment of non-small cell lung cancer. [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 5119.

Abstract 2998: Inhibition of a small GTPase in tumor cells enhances antigen presentation and tumor cell killing in breast cancer

Abstract Breast cancer is the leading cause of cancer in women and the second leading cause of death in female patients. Immune checkpoint blockade (ICB) therapies have shown promise in treating breast cancer patients but only a small proportion of patients respond to ICB therapy partly due to lack of immune cell infiltration into the tumor. Reduced immune infiltration is a result of downregulation of antigen presentation in the tumor cell by decreasing the levels of antigen-MHC-I (major histocompatibility complex-I) molecules on the tumor cell surface. To investigate pathways upregulated in breast cancer, we performed a paradigm tool analysis (Broad Institute) of RNA expression data using TCGA breast invasive carcinoma datasets and found that signaling events of a small GTPase (SGX) ranked 8th among upregulated pathways. SGX has been implicated in the internalization of MHC-I molecules into the cell. To investigate the effects of SGX on antigen presentation, we generated SGX knockdown EO771 and 4T1 murine and MDA-MB-231 human breast tumor cell lines and measured their antigen presentation by flow cytometry. We observed a significant increase in MHC-I levels upon SGX knockdown. We then performed an antibody internalization assay on SGX knockdown cells where fluorophore-labeled MHC-I antibodies were allowed to internalize for 10 min followed by early endosome isolation. There was a significant reduction of MHC-I antibody-containing early endosomes in SGX knockdown cells. Using a commercially available SGX inhibitor, we tested the pharmacological inhibition of SGX and observed a dose-dependent increase in MHC-I MFI. If a SGX inhibitor was to be administered in vivo, we would need to test the impact of inhibitor treatment on CD8+ T cells. To this end, we isolated CD8+ T cells from a C57BL/6 mouse and pretreated them with the inhibitor, activated them in vitro and measured their functionality. Interestingly, inhibitor treatment led to higher IFNg, TNFa and IL-2 production. To explore the possibility of dual SGX inhibition in tumor and T cells, we performed a T cell cytotoxicity assay using EO771 tumor cells and autologous CD8+ T cells and observed increased tumor cell killing when either the tumor cells or T cells were treated with inhibitor. Collectively, we show here that dual SGX inhibition in tumor cells and CD8+ T cells will enhance tumor cell killing due to enhanced antigen presentation and T cell functionality respectively. These results give us more insight into the use of SGX inhibitors in combination with ICB, thus enhancing treatment efficacy. Citation Format: Ishara Moulana, Samantha Sharma, Xinna Zhang, Xiongbin Lu. Inhibition of a small GTPase in tumor cells enhances antigen presentation and tumor cell killing in breast cancer [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 2998.

Abstract 4452: FASN inhibition overcomes immune evasion by upregulating MHC-I in hepatocellular carcinoma

Abstract Immune evasion is one of the important factors leading to the progression of hepatocellular carcinoma(HCC). Restricted antigen presentation by decrease of MHC-I remains a huge barrier to triggering an immune attack. However, knowledge is limited on how to enhance antigen presentation of cancer cells. Here we identify that FASN inhibition by shRNA and inhibitors could boost MHC-I level on HCC cell surface. FASN deficiency also promotes antigen presentation and augments CD8+ T cell cytotoxicity. Mechanistically, FASN knock-down decreases the degradation of MHC-I in lysosomes. Moreover, ZDHHC3-mediated palmitoylation participates in this process. In vivo, orthotopic HCC mouse model shows that Fasn knock-down greatly improves the cytotoxicity of tumor-infiltrating CD8+ T cells and suppresses HCC tumor growth, indicating that inhibiting FASN is a promising way to enhance the efficiency of immunotherapy in HCC. Citation Format: Jiao Huang, Wai Ying Tsang, Xiaona Fang, Yu Zhang, Jie Luo, Lanqi Gong, Baifeng Zhang, Ching Ngar Wong, Beilei Liu, Jinlin Huang, Yuma Yang, Shan Liu, Zhihong Li, Xinyuan Guan. FASN inhibition overcomes immune evasion by upregulating MHC-I in hepatocellular carcinoma. [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 4452.

Abstract 4898: Imipridones and EZH2 inhibitors induce similar changes in cytokines and regulated genes in GBM and DMG while vorinostat potentiates anti-tumor efficacy despite variability in cytokine profiles

Abstract TIC10/ONC201 and ONC206 are small molecule imipridones with anti-cancer activity. ONC201, EZH2i and HDACi can modulate tumor microenvironment (TME) and antitumor immunity. We studied the impact of these drugs or their combinations on the TME or tumor immunity by investigating cytokine profiles. We treated U251 GBM and HCT116 CRC cells with vorinostat and found upregulation of CXCL11, CXCL14, INF-γ and TRAIL, and downregulation of prolactin, CXCL9, VEGF and CCL2 in U251. CXCL11 promotes anti-tumor immunity by increasing activated CD8+ T cells in tumors but was associated with metastasis of GBM. CXCL13 induces tertiary lymphoid structures and enhances infiltration of CD8+ T cells in tumors. INF-γ enhances antigen presentation and promotes activation of NK cells. Vorinostat upregulated the secretion of pro-immune cytokines and TRAIL which induces tumor apoptosis. Downregulated cytokines in U251 were prolactin, CXCL9 involved in tumor growth and metastasis, VEGF, and CCL2 involved in immunosuppression in GBM. Cytokines upregulated in HCT116 included IL-8 contributing to CRC progression, and CXCL13, and CXCL11 which correlates with anti-tumor immunity in colon cancer. Downregulated cytokines in HCT116 were soluble TRAIL R2 functioning as a decoy receptor for TRAIL, VEGF, and prolactin. We treated DMG, GBM and HCC cells with imipridones, EHZ2i tazemetostat or HDACi panobinostat alone or combination of imipridones plus tazemetostat or panobinostat or the triple combination of imipridone plus tazemetostat and panobinostat. We observed that cytokines impairing immunity were downregulated and cytokines promoting immunity were upregulated by individual drugs or combinations. Hep3B HCC cells showed the most robust changes among the mentioned tumors with respect to the changes of cytokine profile following the treatments. Imipridone or EZH2i treated cells showed similar cytokine profile changes in tumor cells. Imipridones downregulated EZH1 and EZH2 proteins in tumor cells. We RNA-seq to investigate gene expression profiles following treatment with ONC201 or tazemetostat. In GBM and DMG, ONC201 and tazemetostat shared similar top regulated genes. GO enrichment analysis showed overlap of top regulated pathways between ONC201 and EZH2i treated cells. Shared regulated pathways in U251 included cell cycle arrest, cell adhesion, nervous system development, cell proliferation, negative regulation of proliferation, PERK-mediated unfolded protein response, extracellular matrix organization, regulation of transcription from RNA polymerase II promoter, and cellular response to hypoxia pathways. We conclude that imipridones ONC201, ONC206, and ONC212 which reduce EZH1 and EZH2 proteins share similar cytokine alterations, gene expression targets and actions with EZH2 inhibitors. Citation Format: Yiqun Zhang, Kelsey Huntington, Wafik S. El-Deiry. Imipridones and EZH2 inhibitors induce similar changes in cytokines and regulated genes in GBM and DMG while vorinostat potentiates anti-tumor efficacy despite variability in cytokine profiles. [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 4898.

Tumor and immune remodeling following radiotherapy in human renal cell carcinoma

BackgroundStudies evaluating peripheral patient samples show radiation can modulate immune responses, yet the biological changes in human tumors particularly at the cellular level remain largely unknown. Here, we address how...

Computer-Aided Discovery of Potent Broad-Spectrum Vaccine Adjuvants.

Adjuvants stimulate the immune system to vigorously respond to a vaccine. While current adjuvants such as aluminum salts and oil-in-water emulsions have been used for decades, they do not generate broad and long-lasting responses in many vaccines. Consequently, more potent adjuvants are needed. Here, using computer-aided molecule design and machine learning, we discovered 2 new, broad-spectrum adjuvants that can boost vaccine responses. Our library containing 46 toll-like receptor (TLR)-targeting agonist ligands were assembled on Au nanoparticles. Comprehensive in vitro, ex vivo and in vivo studies showed both leads promoted dendritic cell activation via multiple TLRs and enhanced antigen presentation to T cells. When used together with tumor-specific antigens to immunize mice against B16-OVA melanoma and 4T1-PD1 breast cancer, both adjuvants unleashed strong immune responses that suppressed tumor growth and lung metastases. Our results show computer-aided design and screening can rapidly uncover potent adjuvants for tackling waning immunity in current vaccines.

Computer‐Aided Discovery of Potent Broad‐Spectrum Vaccine Adjuvants

AbstractAdjuvants stimulate the immune system to vigorously respond to a vaccine. While current adjuvants such as aluminum salts and oil‐in‐water emulsions have been used for decades, they do not generate broad and long‐lasting responses in many vaccines. Consequently, more potent adjuvants are needed. Here, using computer‐aided molecule design and machine learning, we discovered 2 new, broad‐spectrum adjuvants that can boost vaccine responses. Our library containing 46 toll‐like receptor (TLR)‐targeting agonist ligands were assembled on Au nanoparticles. Comprehensive in vitro, ex vivo and in vivo studies showed both leads promoted dendritic cell activation via multiple TLRs and enhanced antigen presentation to T cells. When used together with tumor‐specific antigens to immunize mice against B16‐OVA melanoma and 4T1‐PD1 breast cancer, both adjuvants unleashed strong immune responses that suppressed tumor growth and lung metastases. Our results show computer‐aided design and screening can rapidly uncover potent adjuvants for tackling waning immunity in current vaccines.

Checkpoint kinase 1/2 inhibition potentiates anti-tumoral immune response and sensitizes gliomas to immune checkpoint blockade

Whereas the contribution of tumor microenvironment to the profound immune suppression of glioblastoma (GBM) is clear, tumor-cell intrinsic mechanisms that regulate resistance to CD8 T cell mediated killing are less understood. Kinases are potentially druggable targets that drive tumor progression and might influence immune response. Here, we perform an in vivo CRISPR screen to identify glioma intrinsic kinases that contribute to evasion of tumor cells from CD8 T cell recognition. The screen reveals checkpoint kinase 2 (Chek2) to be the most important kinase contributing to escape from CD8 T-cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are currently being evaluated in clinical trials, in combination with PD-1 or PD-L1 blockade, lead to survival benefit in multiple preclinical glioma models. Mechanistically, loss of Chek2 enhances antigen presentation, STING pathway activation and PD-L1 expression in mouse gliomas. Analysis of human GBMs demonstrates that Chek2 expression is inversely associated with antigen presentation and T-cell activation. Collectively, these results support Chek2 as a promising target for enhancement of response to immune checkpoint blockade therapy in GBM.

Adrenergic receptor signaling regulates the CD40-receptor mediated anti-tumor immunity.

Anti-CD40 agonistic antibody (αCD40), an activator of dendritic cells (DC) can enhance antigen presentation and activate cytotoxic T-cells against poorly immunogenic tumors. However, cancer immunotherapy trials also suggest that αCD40 is only moderately effective in patients, falling short of achieving clinical success. Identifying factors that decrease αCD40 immune-stimulating effects can aid the translation of this agent to clinical reality. Here, we reveal that β-adrenergic signaling on DCs directly interferes with αCD40 efficacy in immunologically cold head and neck tumor model. We discovered that β-2 adrenergic receptor (β2AR) activation rewires CD40 signaling in DCs by directly inhibiting the phosphorylation of IκBα and indirectly by upregulating levels of phosphorylated-cAMP response element-binding protein (pCREB). Importantly, the addition of propranolol, a pan β-Blocker reprograms the CD40 pathways, inducing superior tumor regressions, increased infiltration of cytotoxic T-cells, and a reduced burden of regulatory T-cells in tumors compared to monotherapy. Thus, our study highlights an important mechanistic link between stress-induced β2AR signaling and reduced αCD40 efficacy in cold tumors, providing a new combinatorial approach to improve clinical outcomes in patients.

Evolution and Progress of mRNA Vaccines in the Treatment of Melanoma: Future Prospects.

mRNA vaccines encoding tumor antigens may be able to sensitize the immune system of the host against cancer cells, enhancing antigen presentation and immune response. Since the breakout of the COVID19 pandemic, interest in mRNA vaccines has been accelerating, as vaccination against the virus served as a measure to limit disease spread. Given that immunotherapy has been the cornerstone of melanoma treatment over the last several decades, further innate immunity enhancement by targeted mRNA vaccines could be the next pivotal achievement in melanoma treatment. Preclinical data coming from murine cancer models have already provided evidence of mRNA vaccines' ability to induce host immune responses against cancer. Moreover, specific immune responses have been observed in melanoma patients receiving mRNA vaccines, while the recent KEYNOTE-942 trial may establish the incorporation of the mRNA-4157/V940 vaccine into the melanoma treatment algorithm, in combination with immune checkpoint inhibition. As the existing data are further tested and reviewed, investigators are already gaining enthusiasm about this novel, promising pathway in cancer therapy.

Brain endothelial cells exposure to malaria parasites links type I interferon signalling to antigen presentation, immunoproteasome activation, endothelium disruption, and cellular metabolism.

Cerebral malaria (CM) lethality is attributable to induction of brain edema induction but the cellular mechanisms involving brain microvascular endothelium in CM pathogenesis are unexplored. Activation of the STING-INFb-CXCL10 axis in brain endothelial cells (BECs) is a prominent component of the innate immune response in CM development in mouse models. Using a T cell-reporter system, we show that Type 1 IFN signaling in BECs exposed to Plasmodium berghei-infected erythrocytes (PbA-IE), functionally enhances MHC Class-I antigen presentation through gamma-interferon independent immunoproteasome activation and impacted the proteome functionally related to vesicle trafficking, protein processing/folding and antigen presentation. In vitro assays showed that Type 1 IFN signaling and immunoproteasome activation are also involved in the dysfunction of the endothelial barrier through disturbing gene expression in the Wnt/ß-catenin signaling pathway. We demonstrate that IE exposure induces a substantial increase in BECs glucose uptake while glycolysis blockade abrogates INFb secretion impairing immunoproteasome activation, antigen presentation and Wnt/ß-catenin signaling. Metabolome analysis show that energy demand and production are markedly increased in BECs exposed to IE as revealed by enriched content in glucose and amino acid catabolites. In accordance, glycolysis blockade in vivo delayed the clinical onset of CM in mice. Together the results show that increase in glucose uptake upon IE exposure licenses Type 1 IFN signaling and subsequent immunoproteasome activation contributing to enhanced antigen presentation and impairment of endothelial barrier function. This work raises the hypothesis that Type 1 IFN signaling-immunoproteasome induction in BECs contributes to CM pathology and fatality (1) by increasing antigen presentation to cytotoxic CD8+ T cells and (2) by promoting endothelial barrier dysfunction, that likely favor brain vasogenic edema.

Abstract PD17-07: Cell-Cycle Inhibition and Immune Microenvironment in HR+/HER2- Breast Cancer During and After preoperative ribociclib and letrozole vesus chemotherapy: A correlative analysis of the 1402-SOLTI/CORALLEEN phase 2 trial

Abstract Background Hormone receptor–positive/HER2-negative (HR+/HER2-) breast cancer (BC) is associated with low % of stromal tumor-infiltrating lymphocytes (sTIL) and immune gene expression and poor response to immune checkpoint inhibitors. Evaluating the effect of letrozole and ribociclib (L+R) on the immune microenvironment may suggest new opportunities for immunotherapy-based approaches for HR+/HER2- BCs. Here, we present an exploratory correlative analysis from CORALLEEN, a trial that evaluated the efficacy of L+R (vs. chemotherapy [CHT]) in patients with high-risk PAM50 Luminal B BC (Prat et al. Lancet Oncology. 2022). Methods CORALLEEN is a randomized exploratory study in postmenopausal women with operable stage I-III breast cancer, HR+/HER2- and Luminal B by Prosigna®. Patients were randomized 1:1 to receive either 6 cycles of ribociclib (600mg; 3-weeks-on/1-week-off) plus daily letrozole or CHT: 4 cycles of AC followed by 12 doses of weekly paclitaxel. The primary endpoint was the rate of PAM50 Risk of Relapse (ROR)-low score at surgery in each arm. Samples were prospectively collected at baseline, day 15, and surgery. sTILs score, ki67 IHC and gene expression analysis were determined in all available samples. Complete cell cycle arrest (CCCA) was defined as Ki67≤2.7%. Gene expression profiling by mRNA sequencing (RNAseq) was evaluated. We applied a collection of 194 immune- gene expression signatures (iGES), representing multiple biological pathways and cell types, including. Results 106 patients were randomly assigned to receive neoadjuvant L+R (n=52) or CTH (n=54). Overall, Ki67, sTILs and RNA-seq was available in 95.4%, 96.7% and 83.1% of the samples across the 3 time-points. In terms of cell-cycle inhibition, L+R achieved a significant decrease in Ki67 protein expression and led to higher rates of CCCA at 2 weeks (89.6% vs. 43.2%, p< 0.001) and surgery (45.9% vs 25.5%, P=0.054) compared with CHT. Interestingly, the 11-gene PAM50 proliferative score was significantly lower in tumors with CCAA than in those with non-CCCA (p< 0.001) after L+R, but not after CTH (p = 0.682). In contrast, tumors with CCCA after CHT had a significantly lower rate of tumor cellularity compared to tumors with non-CCAA (p = 0.002). This was not observed in the L+R arm (p=0.141). Compared to baseline, no clear and significant patterns in % of sTILs were observed at week 2 and surgery. However, % of TILs at surgery in tumors with CCCA after CHT was higher than in tumors with non-CCCA (median 15% versus 1%, p=0.017). This was not observed in the L+R arm (median 1% and 5%, p=0.584). Interestingly, this inverse relationship between immune infiltration and CCAA was further confirmed by RNA- CHT compared to tumors with non-CCCA, whereas 174 (89.7%) of iGES were upregulated (FDR< 5%) in tumors with non-CCCA after L+R compared to tumors with CCCA. Finally, L+R and CTH treatment at week 2 and surgery showed an increase in adaptive immune signatures indicative of activated T-cell and B-cell phenotypes; however, CTH was uniquely associated with increased cytokine signaling, enhanced antigen presentation, dendritic, granulocyte, macrophage and NK cells and decrease in Th17, Th2 and Treg cells. Conclusion In early-stage Luminal B breast cancer, L+R induce a potent anti-proliferative effect compared to CHT. Both treatments generally increased T- and B-cell immune infiltration; however, an inverse relationship between immune infiltration and anti-proliferative response at surgery exists according to treatment, where immune infiltration is increased in residual tumors with non-CCAA when treated with L+R, but the opposite is observed with CHT. The prognostic value of immune and anti-proliferative effects of L+R in residual tumors is currently being evaluated in the prospective RIBOLARIS phase II clinical trial (NCT05296746). Citation Format: Tomás Pascual, Nuria Chic, Aranzazu Fernandez-Martinez, Blanca González-Farré, Laia Paré, Cristina Saura, Cristina Hernando, Montserrat Muñoz, Míriam Arumí, Patricia Galván, Xavier Gonzalez-Farré, Mafalda Oliveira, Miguel Gil Gil, Eva Ciruelos, Patricia Villagrasa, Joaquin Gavila Gregori, Aleix Prat, Charles M. Perou. Cell-Cycle Inhibition and Immune Microenvironment in HR+/HER2- Breast Cancer During and After preoperative ribociclib and letrozole vesus chemotherapy: A correlative analysis of the 1402-SOLTI/CORALLEEN phase 2 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-07.

TFAM deficiency in dendritic cells leads to mitochondrial dysfunction and enhanced antitumor immunity through cGAS-STING pathway

BackgroundMitochondrial transcription factor A (TFAM) is a transcription factor that maintains mitochondrial DNA (mtDNA) stabilization and initiates mtDNA replication. However, little is known about the immune regulation function and TFAM...

Laser-assisted intradermal delivery of a microparticle vaccine for respiratory syncytial virus induces a robust immune response.

Respiratory syncytial virus (RSV) is an infectious disease that poses a significant public health risk in young children. Vaccine studies conducted in the 1960s using an intramuscular injection of formalin-inactivated respiratory syncytial virus (Fi-RSV) resulted in an enhanced respiratory disease and led to the failure of the vaccine. Thus, the virus-like particles (VLP) of the RSV fusion (F) protein was used as the vaccine antigen in this study. The F-VLP was encapsulated in a microparticle (MP) matrix composed of cross-linked bovine serum albumin (BSA) to enhance the antigen presentation and uptake. Moreover, a painless vaccination method would be desirable for an infectious disease that mainly affects young children. Thus, an ablative laser device, Precise Laser Epidermal System (P.L.E.A.S.E), was utilized to create micropores on the skin for vaccine delivery. We observed enhanced antigen presentation of the vaccine microparticles (F-VLP MP) with and without the adjuvant monophosphoryl lipid A (MPL-A) MP in dendritic cells. Consequently, Swiss Webster mice were immunized with the adjuvanted vaccine microparticles using the P.L.E.A.S.E laser to study the in vivo immunogenicity. The immunized mice had high serum immunoglobulin (IgG, IgG2a) levels, indicating a Th1 response. Subsequent analysis of lung homogenates post- RSV challenge revealed high IgA, indicating generation of a mucosal immune response upon intradermal immunization. Flowcytometry analysis showed high CD8+, and CD4+ expression in the lymph node and spleen of the adjuvanted vaccine microparticle immunized mice. Increased expression of interferon gamma (IFN-γ) in the spleen cells further proved Th1 polarized immune response. Finally, an immune plaque assay indicated significantly low lung viral titer in the mice immunized with intradermal adjuvanted vaccine microparticles. Thus, ablative laser-assisted immunization with the F-VLP based adjuvanted vaccine microparticles could be a promising vaccine candidate for RSV.

A root cause analysis to identify the mechanistic drivers of immunogenicity against the anti-VEGF biotherapeutic brolucizumab.

Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.