Cytotoxic T Lymphocyte Activity Research Articles

The Immune Microenvironment in Prostate Cancer: A Comprehensive Review.

Prostate cancer (PCa) is a malignancy with significant immunosuppressive properties and limited immune activation. This immunosuppression is linked to reduced cytotoxic T-cell activity, impaired antigen presentation, and elevated levels of immunosuppressive cytokines and immune checkpoint molecules. Studies demonstrate that cytotoxic CD8+ T cell infiltration correlates with improved survival, while increased regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) are associated with worse outcomes and therapeutic resistance. Th1 cells are beneficial, whereas Th17 cells, producing interleukin-17 (IL-17), contribute to tumor progression. Tumor-associated neutrophils (TANs) and immune checkpoint molecules like PD-1/PD-L1 and TIM-3 are also linked to advanced stages of PCa. Chemotherapy holds promise in converting the "cold" tumor microenvironment (TME) to a "hot" one by depleting immunosuppressive cells and enhancing tumor immunogenicity. This comprehensive review examines the immune microenvironment in PCa, focusing on the intricate interactions between immune and tumor cells in the TME. It highlights how TAMs, Tregs, cytotoxic T cells, and other immune cell types contribute to tumor progression or suppression and how PCa's low immunogenicity complicates immunotherapy. The infiltration of cytotoxic CD8+ T cells and Th1 cells correlates with better outcomes, while elevated Tregs and TAMs promote tumor growth, metastasis, and resistance. TANs and NK cells exhibit dual roles, with higher NK cell levels linked to better prognoses. Immune checkpoint molecules like PD-1, PD-L1, and TIM-3 are associated with advanced disease. Chemotherapy can improve tumor immunogenicity by depleting Tregs and MDSCs, offering therapeutic promise.

The branched N-glycan of PD-L1 predicts immunotherapy responses in patients with recurrent/metastatic HNSCC

Immunotherapy has revolutionized cancer treatment, but the lack of a reliable predictive biomarker for treatment response remains a challenge. Alpha-1,6-Mannosylglycoprotein 6-β-N-Acetylglucosaminyltransferase 5 (MGAT5) is a key regulator of complex N-glycan synthesis, and its dysregulation is associated with cancer progression. The lectin Phaseolus vulgaris leukoagglutinin (PHA-L) specifically binds to mature MGAT5 products. Previous studies have indicated elevated PHA-L staining in head and neck squamous cell carcinoma (HNSCC), which implies increased activity of MGAT5. However, the specific role of MGAT5 in HNSCC remains unclear. In this study, we found significantly higher PHA-L staining and MGAT5 expression in HNSCC tumors compared to adjacent non-tumor tissues. Using a mass spectrometry (MS)-based glycoproteomic approach, we identified 163 potential protein substrates of MGAT5. Functional analysis revealed that protein substrates of MGAT5 regulated pathways related to T cell proliferation and activation. We further discovered that PD-L1 was among the protein substrates of MGAT5, and the expression of MGAT5 protected tumor cells from cytotoxic T lymphocyte (CTL) killing. Treatment of nivolumab alleviated the protective effects of MGAT5 on CTL activity. Consistently, patients with MGAT5-positive tumors showed improved responses to immunotherapy compared to those with MGAT5-negative tumors. Using purified PD-L1 from HNSCC cells and a glycoproteomic approach, we further deciphered that the N35 and N200 sites carry the majority of complex N-glycans on PD-L1. Our findings highlight the critical role of MGAT5-mediated branched N-glycans on PD-L1 in modulating the interaction with the immune checkpoint receptor PD-1. Consequently, we propose that MGAT5 could serve as a biomarker to predict patients’ responses to anti-PD-1 therapy. Furthermore, targeting the branched N-glycans at N35 and N200 of PD-L1 may lead to the development of novel diagnostic and therapeutic approaches.

Targeting CDCP1 boost CD8+ T cells-mediated cytotoxicity in cervical cancer via the JAK/STAT signaling pathway

BackgroundCervical cancer remains a global health challenge. The identification of new immunotherapeutic targets may provide a promising platform for advancing cervical cancer treatment.ObjectiveThis study aims to investigate the role of...

A combination of protein phosphatase 2A inhibition and checkpoint immunotherapy: a perfect storm.

Immune checkpoint blockade has emerged as a potent new tool in the war on cancer. However, only a subset of cancer patients benefit from this therapeutic modality, sparking a search for combination therapies to increase the fraction of responding patients. We argue here that inhibition of protein phosphatase 2A (PP2A) is a promising approach to increase responses to immune checkpoint blockade and other therapies that rely on the presence of tumor-reactive Tcells. Inhibition of PP2A increases neoantigen expression on tumor cells, activates the cGAS/STING pathway, suppresses regulatory Tcells, and increases cytotoxic Tcell activation. In preclinical models, inhibition of PP2A synergizes with immune checkpoint blockade and emerging evidence indicates that patients who have tumors with mutations in PP2A respond better to immune checkpoint blockade. Therefore, inhibition of PP2A activity may be an effective way to sensitize cancer cells to immune checkpoint blockade and cell-based therapies using tumor-reactive Tcells.

The TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial

Functional blockade of the transforming growth factor-beta (TGFβ) signalling pathway improves the efficacy of cytotoxic and immunotherapies. Here, we conducted a phase 1b study (ClinicalTrials.gov., NCT03143985) to determine the primary endpoints of safety, tolerability, and maximal tolerated dose (200 mg twice daily) for the orally-available TGFβ type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed and/or refractory multiple myeloma (RRMM) patients who had received ≥2 lines of chemoimmunotherapy. Secondary endpoints demonstrated sustained clinical responses, favorable pharmacokinetic parameters and a 6-month progression-free survival of 82%. Vactosertib combined with pomalidomide was well-tolerated at all dose levels and displayed a manageable adverse event profile. Exploratory analysis indicated that vactosertib co-treatment with pomalidomide also reduced TGFβ levels in patient bone marrow as well as the level of CD8+ T-cells that expressed the immunoinhibitory marker PD-1. In vitro experiments indicated that vactosertib+pomalidomide co-treatment decreased the viability of MM cell lines and patient tumor cells, and increased CD8+ T-cell cytotoxic activity. Vactosertib is a safe therapeutic that demonstrates tumor-intrinsic activity and can overcome immunosuppressive challenges within the tumor microenvironment to reinvigorate T-cell fitness. Vactosertib offers promise to improve immunotherapeutic responses in heavily-pretreated MM patients refractory to conventional agents.

An observational study on the relationship between tyrosine kinase inhibitor treatment-free remission and cytotoxic T-lymphocytes in patients with chronic phase chronic myeloid leukemia.

171 Background: Since the introduction of tyrosine kinase inhibitors (TKIs), the effectiveness of treating chronic phase chronic myeloid leukemia has seen remarkable improvement. A current challenge in TKI therapy is achieving treatment-free remission (TFR) safely. Numerous TKI-stop trials and translational studies have investigated factors influencing TFR, including TKI treatment duration, duration of deep molecular response (DMR), cellular immunity activation, and bcr::abl1 transcript type. However, identifying reliable markers for achieving TFR safely remains uncertain. In this multicenter study, we focused on examining the association between TFR and cytotoxic T-lymphocytes (CTLs). Methods: Between June 2020 and March 2022, a total of 45 patients were enrolled: 38 patients with DMR for at least 1 year on TKIs (on TKI group) and 7 patients with DMR for at least 1 year after discontinuing TKIs (off TKI group). The study included 30 males, with a median age of 59 years (range: 29–87). Median TKI treatment duration was 7.7 years (range: 1.5–18.3), and median TKI cessation duration in the off TKI group was 4.9 years (range: 1.7–7.1). Molecular response and cellular immunity were monitored over 12 months post-consent for patients in the off TKI group and after TKI discontinuation for patients in the on TKI group. Results: During the observation period, 25 patients (65.8%) in the on TKI group maintained DMR, while 13 patients (34.2%) lost DMR. Conversely, all patients in the off TKI group sustained DMR. The median TKI treatment duration in the group maintaining DMR (9.45 years, range: 3.3–18.3, N = 32) significantly exceeded that in the group losing DMR (4.9 years, range: 1.5–11.9, N = 13) (P = 0.0048). Patients taking TKIs for over 7 years with a higher percentage of total memory CTLs than total effector CTLs maintained DMR. Conversely, patients losing DMR despite over 10 years of TKI treatment had a higher percentage of total effector CTLs than total memory CTLs. Among the 32 patients maintaining DMR, 19 exhibited a higher percentage of total effector CTLs than total memory CTLs, with 15 (78.9%) maintaining MR5 throughout the study. Furthermore, of the six patients maintaining DMR with relatively short TKI administration (median 5.5 years, range: 3.3–6.4), three displayed a higher percentage of total memory CTLs than total effector CTLs. Among the remaining three patients, two showed strong CTL activation, with one patient having an effector CTL clone over 30% and the other having a memory CTL clone over 30%. Conclusions: This study indicates that a sufficiently prolonged TKI treatment duration (>7 years) and maintaining deeper DMR (≥ MR4.5, preferably MR5) are conducive to safely achieving TFR. Furthermore, a high percentage of total memory CTLs and intense CTL activation may serve as meaningful markers for TFR.

TLR7 agonist, DSP-0509, with radiation combination therapy enhances anti-tumor activity and modulates T cell dependent immune activation

BackgroundTLR7 is a key player in the antiviral immunity. TLR7 signaling activates antigen-presenting cells including DCs and macrophages. This activation results in the adaptive immunity including T cells and B cells. Therefore, TLR7 is an important molecule of the immune system. Based on these observations, TLR7 agonists considered to become a therapy weaponize the immune system against cancer. Radiation therapy (RT) is one of the standard cancer therapies and is reported to modulate the tumor immune response. In this study, we aimed to investigate the anti-tumor activity in combination of TLR7 agonist, DSP-0509, with RT and underlying mechanism.ResultWe showed that anti-tumor activity is enhanced by combining RT with the TLR7 agonist DSP-0509 in the CT26, LM8, and 4T1 inoculated mice models. We found that once- weekly (q1w) dosing of DSP-0509 rather than biweekly (q2w) dosing is needed to achieve superior anti-tumor activities in CT26 model. Spleen cells from the mice in RT/DSP-0509 combination treatment group showed increased tumor lytic activity, inversely correlated with tumor volume, as measured by the chromium-release cytotoxicity assay. We also found the level of cytotoxic T lymphocytes (CTLs) increased in the spleens of completely cured mice. When the mice completely cured by combination therapy were re-challenged with CT26 cells, all mice rejected CT26 cells but accepted Renca cells. This rejection was not observed with CD8 depletion. Furthermore, levels of splenic effector memory CD8 T cells were increased in the combination therapy group. To explore the factors responsible for complete cure by combination therapy, we analyzed peripheral blood leukocytes (PBLs) mRNA from completely cured mice. We found that Havcr2low, Cd274low, Cd80high, and Il6low were a predictive signature for the complete response to combination therapy. An analysis of tumor-derived mRNA showed that combination of RT and DSP-0509 strongly increased the expression of anti-tumor effector molecules including Gzmb and Il12.ConclusionThese data suggest that TLR7 agonist, DSP-0509, can be a promising concomitant when used in combination with RT by upregulating CTLs activity and gene expression of effector molecules. This combination can be an expecting new radio-immunotherapeutic strategy in clinical trials.

Carrier-Free Photodynamic Bioregulators Inhibiting Lactic Acid Efflux Combined with Immune Checkpoint Blockade for Triple-Negative Breast Cancer Immunotherapy.

Abnormal tumor metabolism creates a complex tumor immune microenvironment that plays a dominant role in the metastasis of triple-negative breast cancer (TNBC). TNBC is insensitive to immune checkpoint blockade (ICB) therapy because of insufficient cytotoxic T lymphocyte (CTL) infiltration and a hyper-lactic acid-suppressive immune microenvironment caused by abnormal glycolysis. Herein, we propose an amplified strategy based on lactic acid regulation to reprogram the immunosuppressive tumor microenvironment (ITM) and combine it with ICB therapy to achieve enhanced antitumor immunotherapy effects. Specifically, we constructed CASN, a carrier-free photodynamic bioregulator, through the self-assembly of the photosensitizer Chlorin e6 and monocarboxylate transporter 1 (MCT1) inhibitor AZD3965. CASN exhibited a uniform structure, good stability, and drug accumulation at the tumor site. CASN-mediated photodynamic therapy following laser irradiation inhibited primary tumor growth and induced immunogenic cell death. Furthermore, CASN reduced lactic acid-mediated regulatory T cell generation and M2 tumor-associated macrophage polarization by blocking MCT1-mediated lactic acid efflux to attenuate immune suppression, inducing the recruitment and activation of CTLs. Ultimately, CASN-mediated immunopotentiation combined with ICB therapy considerably strengthened tumor immunotherapy and effectively inhibited tumor growth and metastasis of TNBC. This synergistic amplification strategy overcomes the limitations of an acidic ITM and presents a potential clinical treatment option for metastatic tumors.

Precursor B Cell Acute Lymphoblastic Leukemia Presenting with Repeated Episodes of Hemophagocytic Lymphohistiocytosis

AbstractHemophagocytic lymphohistiocytosis (HLH) is a rare disorder characterized by dysregulated activation of cytotoxic T-lymphocytes and macrophages, resulting in excessive cytokine release and tissue damage. Although hematological malignancies and the chemotherapies used to treat them are frequently identified as triggering factors for HLH, B lymphoid leukemias are rarely implicated. In this report, we present an interesting case of a patient who presented with symptoms of HLH and was subsequently diagnosed with B-cell acute lymphoblastic leukemia. The further course of chemotherapy was complicated by another episode of HLH. This case highlights the complexity and the diverse triggers of HLH in the context of B lymphoid leukemia. Recognizing this atypical presentation is important to institute timely management strategies.

Oxidized Melanoma Antigens Promote Activation and Proliferation of Cytotoxic T-Cell Subpopulations.

Increasing evidence suggests the role of reactive oxygen and nitrogen species (RONS) in regulating antitumor immune effects and immunosuppression. RONS modify biomolecules and induce oxidative post-translational modifications (oxPTM) on proteins that can alarm phagocytes. However, it is unclear if and how protein oxidation by technical means could be a strategy to foster antitumor immunity and therapy. To this end, cold gas plasma technology producing various RONS simultaneously to oxidize the two melanoma-associated antigens MART and PMEL is utilized. Cold plasma-oxidized MART (oxMART) and PMEL (oxPMEL) are heavily decorated with oxPTMs as determined by mass spectrometry. Immunization with oxidized MART or PMEL vaccines prior to challenge with viable melanoma cells correlated with significant changes in cytokine secretion and altered T-cell differentiation of tumor-infiltrated leukocytes (TILs). oxMART promoted the activity of cytotoxic central memory T-cells, while oxPMEL led to increased proliferation of cytotoxic effector T-cells. Similar T-cell results are observed after incubating splenocytes of tumor-bearing mice with B16F10 melanoma cells. This study, for the first time, provides evidence of the importance of oxidative modifications of two melanoma-associated antigens in eliciting anticancer immunity.

Turning Waste into Wealth: A Potent Sono-Immune Strategy Based on Microcystis.

Currently, sonodynamic therapy (SDT) has limited therapeutic outcomes and immune responses, highlighting the urgent need for enhanced strategies that can stimulate robust and long-lasting antitumor effects. Microcystis, a notorious microalga, reveals the possibility of mediating SDT owing to the presence of gas vesicles (GVs) and phycocyanin (PC). Herein, a nontoxic strain of Microcystis elabens (labeled Me) is developed as a novel agent for SDT because it generates O2 under red light (RL) illumination, while GVs and PC act as cavitation nuclei and sonosensitizers, respectively. Moreover, algal debris is released after ultrasound (US) irradiation, which primes the Toll-like receptor pathway to initiate a cascade of immune responses. This sono-immune strategy inhibits CT26 colon tumor growth largely by promoting dendritic cell (DC) maturation and cytotoxic T-cell activation. After combination with the immune checkpoint blockade (ICB), the therapeutic outcome is further amplified, accompanied by satisfactory abscopal and immune memory effects; the similar potency is proven in the "cold" 4T1 triple-negative breast tumor. In addition, Me exhibits good biosafety without significant acute or chronic toxicity. Briefly, this study turns waste into wealth by introducing sono-immunotherapy based on Microcystis that achieved encouraging therapeutic effects on cancer, which is expected to be translated into the clinic.

Phase 2 randomized study of rintatolimod following FOLFIRINOX in patients with locally advanced pancreatic adenocarcinoma.

TPS4214 Background: The immunosuppressive microenvironment of pancreatic ductal adenocarcinoma (PDAC) is a driver of its lethality and resistance to immunotherapy. Strategies which modulate the immunosuppressive milieu have the potential to improve the anti-tumor response and improve outcomes in patients with PDAC. Rintatolimod is a synthetic double-stranded RNA molecule that activates the toll-like receptor 3 (TLR3) present on dendritic cells, B-cells, natural killer cells and pancreatic epithelial cells. TLR3 stimulation may increase cytotoxic T-cell activity in PDAC through dendritic cell maturation, antigen priming and downstream proliferation of cytotoxic T-cells. Twenty-seven patients were treated with rintatolimod in a single center Named Patient Program at Erasmus University Medical Center in Rotterdam between January 2017 and February 2019. All had locally advanced or metastatic PDAC which had not progressed on FOLFIRINOX (median 8 cycles). Maintenance rintatolimod, administered intravenously twice weekly for up to 18 weeks, was generally well tolerated. The most common toxicities were grade 1-2 myalgia (30%), chills (52%) and fatigue (30%); no toxicities ≥ grade 3 were observed. Progression-free and overall survival (from cycle 1 FOLFIRINOX) were 13 and 19 months. Methods: This is a phase 2 randomized, open-label, multicenter clinical trial. The primary objective is to assess the efficacy of rintatolimod compared to observation in subjects with locally advanced PDAC which has not progressed after FOLFIRINOX. Eligible patients have completed at least 4 months of FOLFIRINOX, have no evidence of progressive disease, have measurable disease by RECIST v1.1, Karnofsky Performance Status ≥80 and have adequate organ function. Subjects with resected PDAC or metastatic disease are not eligible. Subjects are randomized 2:1 to rintatolimod vs. observation. Rintatolimod is administered intravenously twice weekly until disease progression in the experimental arm. Cross-sectional imaging is performed every 8 weeks in both arms. The primary endpoint is progression-free survival, as assessed by the investigator. Secondary endpoints include overall survival, objective response rate, duration of response and safety, as assessed by CTCAE v5.0. Exploratory endpoints include quality of life and changes in immune cell composition and T-cell response in the peripheral blood to characterize the immunomodulatory effects of rintatolimod. The planned sample size is 90 subjects. The final analysis will be performed after 64 events (disease progression or death) have been observed. The study is powered to detect a hazard ratio of 0.47 for median PFS with 80% power and significance level of 5%. Clinical trial information: NCT05494697 .

DC-derived CXCL10 promotes CTL activation to suppress ovarian cancer

This study investigates the role of dendritic cells (DCs), with a focus on their CXCL10 marker gene, in the activation of cytotoxic T lymphocytes (CTLs) within the ovarian cancer microenvironment and its impact on disease progression. Utilizing scRNA-seq data and immune infiltration analysis, we identified a diminished DC presence in ovarian cancer. Gene analysis pinpointed CXCL10 as a key regulator in OV progression via its influence on DCs and CTLs. Prognostic analysis and in vitro experiments substantiated this role. Our findings reveal that DC-derived CXCL10 significantly affects CTL activation and proliferation. Reduced CXCL10 levels hinder CTL cytotoxicity, promoting ovarian cancer cell migration and invasion. Experimental studies using animal models have provided further evidence that the capacity of CTLs to suppress tumor development is significantly diminished when treated with DCs that have low expression of CXCL10. Dendritic cell-derived CXCL10 emerges as a pivotal factor in restraining ovarian cancer growth and metastasis through the activation of cytotoxic T lymphocytes. This study sheds light on the crucial interplay within the ovarian cancer microenvironment, offering potential therapeutic targets for ovarian cancer treatment.

CCL9/CCR1 axis-driven chemotactic nanovesicles for attenuating metastasis of SMAD4-deficient colorectal cancer by trapping TGF-β

SMAD4 deficiency in colorectal cancer (CRC) is highly correlated with liver metastasis and high mortality, yet there are few effective precision therapies available. Here, we show that CCR1+-granulocytic myeloid-derived suppressor cells (G-MDSCs) are highly infiltrated in SMAD4-deficient CRC via CCL15/CCR1 and CCL9/CCR1 axis in clinical specimens and mouse models, respectively. The excessive TGF-β, secreted by tumor-infiltrated CCR1+-G-MDSCs, suppresses the immune response of cytotoxic T lymphocytes (CTLs), thus facilitating metastasis. Hereby, we develop engineered nanovesicles displaying CCR1 and TGFBR2 molecules (C/T-NVs) to chemotactically target the tumor driven by CCL9/CCR1 axis and trap TGF-β through TGF-β-TGFBR2 specific binding. Chemotactic C/T-NVs counteract CCR1+-G-MDSC infiltration through competitive responding CCL9/CCR1 axis. C/T-NVs-induced intratumoral TGF-β exhaustion alleviates the TGF-β-suppressed immune response of CTLs. Collectively, C/T-NVs attenuate liver metastasis of SMAD4-deficient CRC. In further exploration, high expression of programmed cell death ligand-1 (PD-L1) is observed in clinical specimens of SMAD4-deficient CRC. Combining C/T-NVs with anti-PD-L1 antibody (aPD-L1) induces tertiary lymphoid structure formation with sustained activation of CTLs, CXCL13+-CD4+ T, CXCR5+-CD20+ B cells, and enhanced secretion of cytotoxic cytokine interleukin-21 and IFN-γ around tumors, thus eradicating metastatic foci. Our strategy elicits pleiotropic antimetastatic immunity, paving the way for nanovesicle-mediated precision immunotherapy in SMAD4-deficient CRC.

IL3-Driven T Cell-Basophil Crosstalk Enhances Antitumor Immunity.

Cytotoxic T lymphocytes (CTL) are pivotal in combating cancer, yet their efficacy is often hindered by the immunosuppressive tumor microenvironment, resulting in CTL exhaustion. This study investigates the role of interleukin-3 (IL3) in orchestrating antitumor immunity through CTL modulation. We found that intratumoral CTLs exhibited a progressive decline in IL3 production, which was correlated with impaired cytotoxic function. Augmenting IL3 supplementation, through intraperitoneal administration of recombinant IL3, IL3-expressing tumor cells, or IL3-engineered CD8+ T cells, conferred protection against tumor progression, concomitant with increased CTL activity. CTLs were critical for this therapeutic efficacy as IL3 demonstrated no impact on tumor growth in Rag1 knockout mice or following CD8+ T-cell depletion. Rather than acting directly, CTL-derived IL3 exerted its influence on basophils, concomitantly amplifying antitumor immunity within CTLs. Introducing IL3-activated basophils retarded tumor progression, whereas basophil depletion diminished the effectiveness of IL3 supplementation. Furthermore, IL3 prompted basophils to produce IL4, which subsequently elevated CTL IFNγ production and viability. Further, the importance of basophil-derived IL4 was evident from the absence of benefits of IL3 supplementation in IL4 knockout tumor-bearing mice. Overall, this research has unveiled a role for IL3-mediated CTL-basophil cross-talk in regulating antitumor immunity and suggests harnessing IL3 sustenance as a promising approach for optimizing and enhancing cancer immunotherapy. See related Spotlight, p. 798.

BCL2A1 neoepitope-elicited cytotoxic T lymphocytes are a promising individualized immunotherapy of pancreatic cancer.

Conventional treatments have shown a limited efficacy for pancreatic cancer, and immunotherapy is an emerging option for treatment of this highly fatal malignancy. Neoantigen is critical to improving the efficacy of tumor-specific immunotherapy. The cancer and peripheral blood specimens from an HLA-A0201-positive pancreatic cancer patient were subjected to next-generation sequencing, and bioinformatics analyses were performed to screen high-affinity and highly stable neoepitopes. The activation of cytotoxic T lymphocytes (CTLs) by dendritic cells (DCs) loaded with mutBCL2A111-20 neoepitope targeting a BCL2A1 mutant epitope was investigated, and the cytotoxicity of mutBCL2A111-20 neoepitope-specific CTLs to pancreatic cancer cells was evaluated. The mutBCL2A111-20 neoepitope was found to present a high immunogenicity and induce CTLs activation and proliferation, and these CTLs were cytotoxic to mutBCL2A111-20 neoepitope-loaded T2 cells and pancreatic cancer PANC-1-Neo and A2-BxPC-3-Neo cells that overexpressed mutBCL2A111-20 neoepitopes, appearing to be a targeting neoepitope specificity. In addition, high BCL2A1 expression correlated with a low 5-yr progression-free interval among pancreatic cancer patients. Our findings provide experimental supports to individualized T cell therapy targeting mutBCL2A111-20 neoepitopes, and provide an option of immunotherapy for pancreatic cancer.

In Situ Hydrogel ModulatescDC1-Based Antigen Presentation and Cancer Stemness to Enhance Cancer Vaccine Efficiency.

Effective presentation of antigens by dendritic cells (DC) is essential for achieving a robust cytotoxic T lymphocytes (CTLs) response, in which cDC1 is the key DC subtype for high-performance activation of CTLs. However, low cDC1 proportion, complex process, and high cost severely hindered cDC1 generation and application. Herein, the study proposes an in situ cDC1 recruitment and activation strategy with simultaneous inhibiting cancer stemness for inducing robust CTL responses and enhancing the anti-tumor effect. Fms-like tyrosine kinase 3 ligand (FLT3L), Poly I:C, and Nap-CUM (NCUM), playing the role of cDC1 recruitment, cDC1 activation, inducing antigen release and decreasing tumor cell stemness, respectively, are co-encapsulated in an in situ hydrogel vaccine (FP/NCUM-Gel). FP/NCUM-Gel is gelated in situ after intra-tumoral injection. With the near-infrared irradiation, tumor cell immunogenic cell death occurred, tumor antigens and immunogenic signals are released in situ. cDC1 is recruited to tumor tissue and activated for antigen cross-presentation, followed by migrating to lymph nodes and activating CTLs. Furthermore, tumor cell stemness are inhibited by napabucasin, which can help CTLs to achieve comprehensive tumor killing. Collectively, the proposed strategy of cDC1 in situ recruitment and activation combined with stemness inhibition provides great immune response and anti-tumor potential, providing new ideas for clinical tumor vaccine design.

Abstract 2909: ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms

Abstract CCAAT/Enhancer Binding Protein β (C/EBPβ) is a transcription factor that is an established driver of cellular transformation in several cancer types with poor prognosis, including glioblastoma (GBM), triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC). These cancers’ growth is often supported by an immunosuppressive (IS) tumor microenvironment (TME), characterized by high expression of TAMs, MDSCs, Tregs, and in the case of GBM, IS microglia. In addition to its cell-autonomous role of promoting survival and proliferation in cancer cells, C/EBPβ is also critical for IS M2 macrophage polarization, while its role in other TME populations is less understood. ST101 is a peptide antagonist of C/EBPβ that is being evaluated in a Phase 2 clinical study in patients with recurrent and newly diagnosed glioblastoma (NCT04478279). Here we explored the impact of ST101 on IS TME cell populations and on activation of cytotoxic T-cells in macrophage co-culture systems. ST101 increased the M1:M2 ratio up to 40-fold in hPBMC-derived macrophages cultured under conditions that typically drive M2 polarization. Further, ST101 promoted M2 repolarization to the M1 cell type, demonstrating that C/EBPβ is required for maintenance of the M2 program. When added to co-cultures of T cells with M2 macrophages, ST101 induced a 3-4-fold increase in intracellular IFN-γ staining indicating enhanced T cell activation. Notably, in M1 cultures ST101 further enhanced expression of the immune activity marker CD80 and T cell activation. Similar to its effect on M2 repolarization, ST101 induced activation of iPs-derived IS human microglia cultures toward an M1-like program. ST101 also impacted IS cell populations in vivo, as demonstrated in an orthotopic TNBC model in which ST101 exposure was associated with a reduction of the TAM fraction, increased intratumoral M1/M2 ratios, reduction of Tregs, increase of CD8:Treg fraction, and enhanced activity in combination with anti-PD-1. Similar observations were observed in clinical samples, where following ST101 exposure a significant decrease in expression of factors involved in M2 polarization, including CD209, SIGLEC5, and IL-24, and T cell suppression, including FoxP3 and inhibitory KIR proteins, were observed. Gene expression analysis suggests that ST101 induced a decrease in the intratumoral Treg:TIL ratio, indicating a shift towards a more immune-active TME. Overall, these results provide novel evidence for the contribution of C/EBPβ to the expression of multiple IS cancer-associated cell types and support the use of ST101 to antagonize C/EBPβ and promote an immune-active TME. Further, these data provide rational for evaluating the clinical impact of ST101 in combinations with immune checkpoint inhibitors. Citation Format: Claudio Scuoppo, Julia Diehl, Ricardo Ramirez, Mark Koester, Erin Gallagher, Siok Leong, Jerel Gonzales, Karen Mendelson, Zach Mattes, Lila Ghamsari, Gene Merutka, Abi Vainstein-Haras, Barry J. Kappel, Jim A. Rotolo. ST101, a clinical CEBPβ antagonist peptide, promotes an immune-active tumor microenvironment by multiple cellular mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2909.

Abstract 5359: Targeted engineering mRNA 3′UTR length enhances immunotherapy response in prostate cancer

Abstract Background: Global mRNA 3′untranslated region (3′UTR) shortening through alternative polyadenylation (APA) has been widely observed in most cancers; however, it has not been demonstrated whether targeted interference of specific oncogenic mRNA 3′UTR lengths can inhibit tumor growth and potentiate immunotherapy response in prostate cancer. Methods: DaPars algorithm was applied for de novo identification of dynamic alternative polyadenylation (APA) during prostate cancer progression to the lethal phase; 3′UTR polyadenylation site (PAS) locations and usages were identified by Poly(A)-ClickSeq (PAC-seq) and 3′RACE; APA transcripts were quantified by RHAPA assay; a 3′UTR CRISPR/dCas13 Engineering System (3′UTRCES) was developed to manipulate the length of desired 3′UTRs; RIP-qPCR and PAR-CLIP-qPCR assays were used to determine the mechanistic basis of 3′UTRCES in APA editing; RNA-seq was utilized to evaluate the off-target effects; TT3 Lipid-like nanoparticles (LLN) were applied for intratumoral delivery of 3′UTRCES RNA molecules for targeted 3′UTR therapy. Quantitative proteomics and immunoprecipitation assays were conducted to investigate how major histocompatibility complex class I (MHC-I) substrates were recognized by SPSB1-containing ubiquitin ligase complex for degradation; Flow cytometry and T cell cytotoxicity assays were performed to evaluate MHC-I level and killing of tumor cells by antigen-stimulated CD8+ T cells. Results: 3′UTR globally shortens during prostate cancer progression to castration-resistance. Through blocking the proximal PASs, 3′UTRCES efficiently and specifically reverses the 3′UTR shortening of novel APA-linked, clinically-relevant prostate cancer oncogenic mRNAs, such as SPSB1, leading to reduced SPSB1 mRNA translation and prostate cancer cell proliferation. Intratumoral injection of TT3 LLN encapsulating 3′UTRCES RNA molecules effectively and safely inhibits prostate tumor growth in engrafted and transgenic mouse models. Notably, downregulation of SPSB1 protein by 3′UTRCES disrupts the interactions of SPSB1-containing ubiquitin ligase complex, leading to compromised ubiquitination-mediated MHC-I degradation and increased stability and abundance of MHC-I protein. Consistently, 3′UTRCES enhances MHC-I-regulated antigen presentation and thereby augments CD8+ T cell-mediated cytotoxicity, which suggest the potentials of 3′UTRCES to sensitize prostate cancer to immune checkpoint therapies. Importantly, 3′UTR shortening of SPSB1 mRNA is significantly associated with decreased MHC-I expression, reduced cytotoxic T-cell infiltration and activation in castration resistant prostate cancer patients. Conclusions: Our results establish the concept of “3′UTR targeted therapy” for treatment of prostate cancer with broad applications to other cancers and other 3′UTR-related diseases. Citation Format: Furong Huang, Fuwen Yuan, Ya Cui, Lei Li, Kexin Li, Zhifen Cui, Jingyue Yan, Qiang Chen, Christopher Nicchitta, Wenbin Ye, Yuebao Zhang, William Hankey, Jeffrey Everitt, Ming Chen, Jiaoti Huang, Hongyan Wang, Xin Lu, Eric Wagner, Yizhou Dong, Wei Li, Qianben Wang. Targeted engineering mRNA 3′UTR length enhances immunotherapy response in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5359.

Abstract 6679: Deciphering the mechanism of action of a live biotherapeutic product for the treatment of cancer

Abstract Intestinal microbiota is essential in shaping the immune system and its functions. In our effort to harness the potential of beneficial gut microbiota, we identified specific bacterial species that are less abundant in colorectal cancer patients compared to healthy individuals. Oral supplementation of these bacteria causes an effective anti-tumor activity in various cancer models through specific activation of cytotoxic T-cells. Based on these results, we aimed to identify the mechanism of action of this bacterial treatment. In vivo studies were performed in C57BL/6 and BALB/c mice. MC-38, CT-26, B16, LLC1.1 and 4T1 cells were used for the heterotopic tumor injections. Immunophenotyping was performed using flow cytometry and immunohistochemistry. Metagenomics analysis and whole genome sequence were performed to identify the bacterial strains. Metabolomics analyses were conducted to analyze bacterial supernatant and serum compositions of mice and patients. Since we found that the bacterial treatment led to a systemic anti-tumor effect, we analyzed serum of the mice that received the bacteria and found that a specific metabolite was highly upregulated compared to untreated mice. Analysis of the medium supernatant from cultures of the bacterial strains led to the discovery that metabolite identified in the serum is being produced by our bacteria. Applying this bacterial metabolite alone in the mouse cancer models led to a similar immune activation and anti-tumor effect as the supplementation with our bacterial strains. Furthermore, we identified the receptor that is targeted by this molecule through experiments with known agonists and antagonists for the target. The anti-tumor effect of the bacteria as well as the bacterial metabolite was blocked through the application of a receptor antagonist, proving that activation of this receptor is the mechanism that drives the enhanced anti-tumor immune response. Activation of the receptor, which is mainly expressed on macrophages led to enhanced IL-12 production and recruitment of cytotoxic T-cells into the tumor. Together, we have fully characterized the mechanism of action of our bacterial treatment, including the secreted active metabolite, the target receptor, and the activated immune cells. We are developing a live biotherapeutic product for the treatment of cancer based on oral administration of lyophilized bacteria. The known mechanism of action of our bacteria helps us to optimize the efficacy of our product, define the right dosage and understand possible side effects. Additionally, it enables us to improve patient selection and prediction of treatment response. Since our product is based on commensal bacteria that are commonly present in healthy individuals, we expect a very good safety and tolerability profile. Therefore, our therapy aims to significantly improve the efficacy of cancer treatment as well as the quality of life of cancer patients. Citation Format: Egle Katkeviciute, Francesca Ferraro, Martin Schwill, Philipp Busenhart, Ana Montalban-Arques. Deciphering the mechanism of action of a live biotherapeutic product for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6679.