Tumor Immunotherapy Research Articles

Fatostatin promotes anti-tumor immunity by reducing SREBP2 mediated cholesterol metabolism in tumor-infiltrating T lymphocytes

Aberrant lipid metabolism impacts intratumoral T cell-mediated immune response and tumor growth. Fatostatin functions as an inhibitor of sterol regulatory element binding protein (SREBP) activation. However, the complex effects of fatostatin on cholesterol metabolism in the tumor microenvironment (TME) and its influence on T cell anti-tumor immunity remain unclear. In this study, fatostatin effectively suppressed B16 melanoma, MC38 colon cancer, and Lewis lung cancer (LLC) transplanted tumor growth in immunocompetent mice by reducing SREBPs-mediated lipid metabolism, especially cholesterol levels. Mechanistically, fatostatin decreased intracellular cholesterol accumulation and inhibited X-box binding protein 1 (XBP1)-mediated endoplasmic reticulum (ER) stress, reducing Treg cells and alleviating CD8+ T cell exhaustion in the TME, exerting anti-tumor activity. Nevertheless， this effect was impaired in immunodeficient nude mice, suggesting fatostatin's anti-tumor efficacy in transplanted tumors partly relies on T cell-mediated anti-tumor immunity. Our study highlights SREBP2-mediated cholesterol metabolism as a potential strategy for anti-tumor immunotherapy, and confirms fatostatin's promise in tumor immunotherapy.

Anti-angiogenic agents combined with immunotherapy for advanced non-small cell lung cancer: a systematic review and meta-analysis.

Anti-angiogenic agents could enhance tumor immunity response, and anti-angiogenesis plus immunotherapy has become a novel treatment option for advanced non-small cell lung cancer (NSCLC). The efficacy of this combination therapy remains controversial and obscure. We conducted a meta-analysis to evaluate the clinical efficacy and safety of this therapeutic strategy in patients with advanced NSCLC and provide more guidance for treating NSCLC clinically. A systematic literature search was performed in PubMed, Embase, Web of Science, CNKI, and Wanfang databases to identify relevant studies published up to December 2021. The primary endpoint was the objective response rate (ORR). Second endpoints were progression-free survival (PFS), overall survival (OS), and grade ≥3 AEs adverse events (AEs). The sensitivity analysis was conducted to confirm the stability of the results. STATA 15.0 was utilized for all pooled analyses. Eleven studies were eventually included in the meta-analysis, involving 533 patients with advanced NSCLC. The pooled ORR rate was 27% (95% CI 18% to 35%; I2 =84.2%; p<0.001), while the pooled median PFS and OS was 5.84 months (95% CI 4.66 to 7.03 months; I2=78.4%; p<0.001) and 14.20 months (95% CI 11.08 to 17.32 months; I2=82.2%; p=0.001), respectively. Most common grade ≥3 AEs included hypertension, hand-foot syndrome, diarrhea, adrenal insufficiency, hyponatremia, proteinuria, rash, thrombocytopenia, and fatigue. Anti-angiogenesis combined with immunotherapy demonstrated satisfactory antitumor activity and an acceptable toxicity profile in patients with advanced NSCLC. The pooled results of our meta-analysis provided further evidence supporting the favorable efficacy and safety of this therapeutic strategy.

Advances in Therapeutic Cancer Vaccines, Their Obstacles, and Prospects Toward Tumor Immunotherapy.

Over the past few decades, cancer immunotherapy has experienced a significant revolution due to the advancements in immune checkpoint inhibitors (ICIs) and adoptive cell therapies (ACTs), along with their regulatory approvals. In recent times, there has been hope in the effectiveness of cancer vaccines for therapy as they have been able to stimulate de novo T-cell reactions against tumor antigens. These tumor antigens include both tumor-associated antigen (TAA) and tumor-specific antigen (TSA). Nevertheless, the constant quest to fully achieve these abilities persists. Therefore, this review offers a broad perspective on the existing status of cancer immunizations. Cancer vaccine design has been revolutionized due to the advancements made in antigen selection, the development of antigen delivery systems, and a deeper understanding of the strategic intricacies involved in effective antigen presentation. In addition, this review addresses the present condition of clinical tests and deliberates on their approaches, with a particular emphasis on the immunogenicity specific to tumors and the evaluation of effectiveness against tumors. Nevertheless, the ongoing clinical endeavors to create cancer vaccines have failed to produce remarkable clinical results as a result of substantial obstacles, such as the suppression of the tumor immune microenvironment, the identification of suitable candidates, the assessment of immune responses, and the acceleration of vaccine production. Hence, there are possibilities for the industry to overcome challenges and enhance patient results in the coming years. This can be achieved by recognizing the intricate nature of clinical issues and continuously working toward surpassing existing limitations.

Dual function of activated PPARγ by ligands on tumor growth and immunotherapy.

As one of the peroxisome-proliferator-activated receptors (PPARs) members, PPARγ is a ligand binding and activated nuclear hormone receptor, which is an important regulator in metabolism, proliferation, tumor progression, and immune response. Increased evidence suggests that activation of PPARγ in response to ligands inhibits multiple types of cancer proliferation, metastasis, and tumor growth and induces cell apoptosis including breast cancer, colon cancer, lung cancer, and bladder cancer. Conversely, some reports suggest that activation of PPARγ is associated with tumor growth. In addition to regulating tumor progression, PPARγ could promote or inhibit tumor immunotherapy by affecting macrophage differentiation or T cell activity. These controversial findings may be derived from cancer cell types, conditions, and ligands, since some ligands are independent of PPARγ activity. Therefore, this review discussed the dual role of PPARγ on tumor progression and immunotherapy.

Integration of multiomics analyses reveals unique insights into CD24-mediated immunosuppressive tumor microenvironment of breast cancer.

Tumor immunotherapy brings new light and vitality to breast cancer patients, but low response rate and limitations of therapeutic targets become major obstacles to its clinical application. Recent studies have shown that CD24 is involved in an important process of tumor immune regulation in breast cancer and is a promising target for immunotherapy. In this study, singleR was used to annotate each cell subpopulation after t-distributed stochastic neighbor embedding (t-SNE) methods. Pseudo-time trace analysis and cell communication were analyzed by Monocle2 package and CellChat, respectively. A prognostic model based on CD24-related genes was constructed using several machine learning methods. Multiple quantitative immunofluorescence (MQIF) was used to evaluate the spatial relationship between CD24+PANCK+cells and exhausted CD8+T cells. Based on the scRNA-seq analysis, 1488 CD24-related differential genes were identified, and a risk model consisting of 15 prognostic characteristic genes was constructed by combining the bulk RNA-seq data. Patients weredivided into high- and low-risk groups based on the median risk score. Immune landscape analysis showed that the low-risk group showed higher infiltration of immune-promoting cells and stronger immune reactivity. The results of cell communication demonstrated a strong interaction between CD24+epithelial cells and CD8+T cells. Subsequent MQIF demonstrated a strong interaction between CD24+PANCK+ and exhausted CD8+T cells with FOXP3+ in breast cancer. Additionally, CD24+PANCK+ and CD8+FOXP3+T cells were positively associated with lower survival rates. This study highlights the importance of CD24+breast cancer cells in clinical prognosis and immunosuppressive microenvironment, which may provide a new direction for improving patient outcomes.

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-β and PD-L1 enhances antitumor responses

ABSTRACT T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-β play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-β inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-β, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-β by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.

Identification of a robust biomarker LAPTM4A for glioma based on comprehensive computational biology and experimental verification.

Glioma, a highly invasive and deadly form of human neoplasm, presents a pressing need for the exploration of potential therapeutic targets. While the lysosomal protein transmembrane 4A (LATPM4A) has been identified as a risk factor in pancreatic cancer patients, its role in glioma remains unexplored. The analysis of differentially expressed genes (DEG) was conducted from The Cancer Genome Atlas (TCGA) glioma dataset and the Genotype Tissue Expression (GTEx) dataset. Through weighted gene co-expression network analysis (WGCNA), the key glioma-related genes were identified. Among these, by using Kaplan-Meier (KM) analysis and univariate/multivariate COX methods, LAPTM4A emerged as the most influential gene. Moreover, the bioinformatics methods and experimental verification were employed to analyze its relationships with diagnosis, clinical parameters, epithelial-mesenchymal transition (EMT), metastasis, immune cell infiltration, immunotherapy, drug sensitivity, and ceRNA network. Our findings revealed that LAPTM4A was up-regulated in gliomas and was associated with clinicopathological features, leading to poor prognosis. Furthermore, functional enrichment analysis demonstrated that LATPM4A played a role in the immune system and cancer progression. In vitro experiments indicated that LAPTM4A may influence metastasis through the EMT pathway in glioma. Additionally, we found that LAPTM4A was associated with the tumor microenvironment (TME) and immunotherapy. Notably, drug sensitivity analysis revealed that patients with high LAPTM4A expression were sensitive to doxorubicin, which contributed to a reduction in LAPTM4A expression. Finally, we uncovered the FGD5-AS1-hsa-miR-103a-3p-LAPTM4A axis as a facilitator of glioma progression. In conclusion, our study identifies LATPM4A as a promising biomarker for prognosis and immune characteristics in glioma.

Sulfonium-Stapled Peptides-Based Neoantigen Delivery System for Personalized Tumor Immunotherapy and Prevention.

Neoantigen peptides hold great potential as vaccine candidates for tumor immunotherapy. However, due to the limitation of antigen cellular uptake and cross-presentation, the progress with neoantigen peptide-based vaccines has obviously lagged in clinical trials. Here, a stapling peptide-based nano-vaccine is developed, comprising a self-assembly nanoparticle driven by the nucleic acid adjuvant-antigen conjugate. This nano-vaccine stimulates a strong tumor-specific T cell response by activating antigen presentation and toll-like receptor signaling pathways. By markedly improving the efficiency of antigen/adjuvant co-delivery to the draining lymph nodes, the nano-vaccine leads to 100% tumor prevention for up to 11 months and without tumor recurrence, heralding the generation of long-term anti-tumor memory. Moreover, the injection of nano-vaccine with signal neoantigen eliminates the established MC-38 tumor (a cell line of murine carcinoma of the colon without exogenous OVA protein expression) in 40% of the mice by inducing potent cytotoxic T lymphocyte infiltration in the tumor microenvironment without substantial systemic toxicity. These findings represent that stapling peptide-based nano-vaccine may serve as a facile, general, and safe strategy to stimulate a strong anti-tumor immune response for the neoantigen peptide-based personalized tumor immunotherapy.

Glycyrrhiza polysaccharides may have an antitumor effect in γδT cells through gut microbiota and TLRs/NF-κB pathway in mice.

Tumor immunotherapy can be a suitable cancer treatment option in certain instances. Here we investigated the potential immunomodulatory effect of oral glycyrrhiza polysaccharides (GCP) on the antitumor function of γδTcells in intestinal epithelial cells in mice. We found that GCP can inhibit tumor growth and was involved in the regulation of systemic immunosuppression. GCP administration also promoted the differentiation of gut epithelia γδTcells into IFN-γ-producing subtype through regulation of local cytokines in gut mucosa. GCP administration increased local cytokine levels through gut microbiota and the gut mucosa Toll-like receptors / nuclear factor kappa-B pathway. Taken together, our results suggest that GCP might be a suitable candidate for tumor immunotherapy, although further clinical research, including clinical trials, are required to validate these results.

Prognosis and therapeutic significance of IGF-1R-related signaling pathway gene signature in glioma

BackgroundGlioma is the most common cancer of the central nervous system with poor therapeutic response and clinical prognosis. Insulin-like growth factor 1 receptor (IGF-1R) signaling is implicated in tumor development and progression and induces apoptosis of cancer cells following functional inhibition. However, the relationship between the IGF-1R-related signaling pathway genes and glioma prognosis or immunotherapy/chemotherapy is poorly understood.MethodsLASSO–Cox regression was employed to develop a 16-gene risk signature in the TCGA-GBMLGG cohort, and all patients with glioma were divided into low-risk and high-risk subgroups. The relationships between the risk signature and the tumor immune microenvironment (TIME), immunotherapy response, and chemotherapy response were then analyzed. Immunohistochemistry was used to evaluate the HSP90B1 level in clinical glioma tissue.ResultsThe gene risk signature yielded superior predictive efficacy in prognosis (5-year area under the curve: 0.875) and can therefore serve as an independent prognostic indicator in patients with glioma. The high-risk subgroup exhibited abundant immune infltration and elevated immune checkpoint gene expression within the TIME. Subsequent analysis revealed that patients in the high-risk subgroup benefited more from chemotherapy. Immunohistochemical analysis confirmed that HSP90B1 was overexpressed in glioma, with significantly higher levels observed in glioblastoma than in astrocytoma or oligodendrocytoma.ConclusionThe newly identified 16-gene risk signature demonstrates a robust predictive capacity for glioma prognosis and plays a pivotal role in the TIME, thereby offering valuable insights for the exploration of novel biomarkers and targeted therapeutics.

Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.

Reducing PD-L1 Expression by Degraders and Downregulators as a Novel Strategy to Target the PD-1/PD-L1 Pathway.

Targeting the programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway has evolved into one of the most promising strategies for tumor immunotherapy. Thus far, multiple monoclonal antibody drugs have been approved for treating a variety of tumors, while the development of small-molecule PD-1/PD-L1 inhibitors has lagged far behind, with only a few small-molecule inhibitors entering clinical trials. In addition to antibody drugs and small-molecule inhibitors, reducing the expression levels of PD-L1 has attracted extensive research interest as another promising strategy to target the PD-1/PD-L1 pathway. Herein, we analyze the structures and mechanisms of molecules that reduce PD-L1 expression and classify them as degraders and downregulators according to whether they directly bind to PD-L1. Moreover, we discuss the potential prospects for developing PD-L1-targeting drugs based on these molecules. It is hoped that this perspective will provide profound insights into the discovery of potent antitumor immunity drugs.

Potential application mechanism of traditional Chinese medicine in treating immune checkpoint inhibitor-induced colitis

Cancer ranks among the foremost causes of mortality worldwide, posing a significant threat to human lives. The advent of tumor immunotherapy has substantially transformed the therapeutic landscape for numerous advanced malignancies, notably non-small cell lung cancer and melanoma. However, as immune checkpoint inhibitors (ICIs) are increasingly applied in clinical settings, a spectrum of undesired reactions, termed immune-related adverse events (irAEs), has emerged. These adverse reactions are associated with immunotherapy and can result in varying degrees of harm to the human body. Among these reactions, Immune checkpoint inhibitor-induced colitis (ICIIC) stands out as one of the most prevalent clinical adverse events. In contemporary times, traditional Chinese medicine (TCM) has demonstrated remarkable efficacy in addressing various maladies. Consequently, investigating the potential application and mechanisms of Chinese medicine in countering immune checkpoint inhibitor-induced colitis assumes significant importance in the treatment of this condition.

Ultra‐Galactocation to Sialic Acid on Tumor Cells with A Penta‐Functional Dendritic Probe for Enhanced Immune‐Killing

AbstractGlycans on tumor cell surface have significant impacts in the immune‐killing process. Here an ultra‐galactocation to sialic acid (Sia) strategy is designed to hugely introduce galactose (Gal) to Sia and on tumor cells in vivo by using a penta‐functional dendritic probe (Den@5F), which efficiently enhances the immune‐killing of tumor cells. The Den@5F contains five different kinds of functional groups, including Gal, Cy5, amino, phenylboronic acid (PBA) and 4‐(4‐(hydroxymethyl)‐2‐methoxy‐5‐nitrophenoxy) butanoate (mNB), which can be conveniently prepared through a two‐step reaction. After injecting into the tumor‐bearing mouse, Den@5F can efficiently block Sia through the specific recognition between PBA and Sia on tumor cells and hugely introduce Gal through the subsequent photo‐crosslinking between mNB and amino groups to multiply conjugate excessive Den@5Fs. The comprehensively blocked Sia can prevent the immune escape, and the hugely introduced Gal can promote the immune stimulation of the immune cells, which lead to an efficient enhancement of the immune‐killing. The proposed strategy provides a significant and promising tool to promote the clinical immunotherapy of tumor.

Targeting novel regulated cell death: Ferroptosis, pyroptosis and necroptosis in anti-PD-1/PD-L1 cancer immunotherapy.

Chemotherapy, radiotherapy, and immunotherapy represent key tumour treatment strategies. Notably, immune checkpoint inhibitors (ICIs), particularly anti-programmed cell death 1 (PD1) and anti-programmed cell death ligand 1 (PD-L1), have shown clinical efficacy in clinical tumour immunotherapy. However, the limited effectiveness of ICIs is evident due to many cancers exhibiting poor responses to this treatment. An emerging avenue involves triggering non-apoptotic regulated cell death (RCD), a significant mechanism driving cancer cell death in diverse cancer treatments. Recent research demonstrates that combining RCD inducers with ICIs significantly enhances their antitumor efficacy across various cancer types. The use of anti-PD-1/PD-L1 immunotherapy activates CD8+ T cells, prompting the initiation of novel RCD forms, such as ferroptosis, pyroptosis, and necroptosis. However, the functions and mechanisms of non-apoptotic RCD in anti-PD1/PD-L1 therapy remain insufficiently explored. This review summarises the emerging roles of ferroptosis, pyroptosis, and necroptosis in anti-PD1/PD-L1 immunotherapy. It emphasises the synergy between nanomaterials and PD-1/PD-L1 inhibitors to induce non-apoptotic RCD in different cancer types. Furthermore, targeting cell death signalling pathways in combination with anti-PD1/PD-L1 therapies holds promise as a prospective immunotherapy strategy for tumour treatment.

Editorial: Novel biomarkers in tumor immunity and immunotherapy

Editorial: Novel biomarkers in tumor immunity and immunotherapy

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs)

IntroductionMacrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages.MethodsHE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment.ResultsIn this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What’s more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth.DiscussioniBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

CD74 as a prognostic and M1 macrophage infiltration marker in a comprehensive pan-cancer analysis.

CD74 is a type-II transmembrane glycoprotein that has been linked to tumorigenesis. However, this association was based only on phenotypic studies, and, to date, no in-depth mechanistic studies have been conducted. In this study, combined with a multi-omics study, CD74 levels were significantly upregulated in most cancers relative to normal tissues and were found to be predictive of prognosis. Elevated CD74 expression was associated with reduced levels of mismatch-repair genes and homologous repair gene signatures in over 10 tumor types. Multiple fluorescence staining and bulk, spatial, single-cell transcriptional analyses indicated its potential as a marker for M1 macrophage infiltration in pan-cancer. In addition, CD74 expression was higher in BRCA patients responsive to conventional chemotherapy and was able to predict the prognosis of these patients. Potential CD74-activating drugs (HNHA and BRD-K55186349) were identified through molecular docking to CD74. The findings indicate activation of CD74 may have potential in tumor immunotherapy.

Abstract LB310: Single-cell transcriptomics and TCR clonality reveal a role for immunotherapy in pediatric brain tumors

Abstract Central nervous system tumors are the most common solid tumors and the leading cause of cancer-related deaths in children. Outcomes remain dismal for a significant proportion of patients despite aggressive multimodal therapy and survivors suffer from treatment-related morbidity and neurological deficits. Despite their remarkable success in adult cancers, immunotherapies are not approved for pediatric brain tumors. Several factors like the relatively smaller patient population, a tumor location that makes them difficult to access through biopsies and poorly-studied tumor immune microenvironment (TME) have posed challenges in advancing immunotherapeutic options for these patients. Immune checkpoint blockers (ICB) tested in unstratified cohorts of children with brain tumors have not shown promising results; therefore, the role of immunotherapy remains unclear. Because the magnitude and nature of pre-existing T cell responses in the tumor has been shown to correlate with response to ICB, we asked if pediatric brain tumors show evidence of such favorable T cell responses. In the current study, we address this question using an unbiased approach to analyze the clonal relationship and transcriptional status of T cells present in tumor tissue of children with brain tumors using paired single-cell RNA-sequencing and TCR-sequencing. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with potential to recognize tumor antigens. Such clonally-expanded T cells display states linked to anti-tumor immunity, express higher levels of transcripts encoding for molecules linked to T cell activation, effector functions, immune cell recruitment, tissue-residency, immune checkpoints, and importantly, show significant enrichment of signatures linked to neoantigen-specific T cells and immunotherapy response. Notably, we identify several neoantigens in pediatric brain tumors, and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes in high-grade glioma. We further show that PD1-expressing CD8+ T cells in pediatric brain tumors are indeed functional as evidenced by their capacity for cytotoxicity, cytokine production and proliferation. Among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and expression of transcripts encoding immune checkpoints in tumor-infiltrating T cells. Overall, our findings suggest that accurate characterization of intra-tumoral T cell responses for features linked to immune check point therapy (ICB) response may enable selection of patients where immunotherapy is likely to be beneficial, an approach that requires prospective validation in future clinical trials. Citation Format: Anusha Preethi Ganesan, Aditi Upadhye, Kevin Meza Landeros, Ciro Ramírez-Suástegui1, Benjamin Schmiedel, Edwin Woo, Serena J. Chee, Denise Malicki, Nicole Coufal, David Gonda, Michael Levy, Jason Greenbaum, Gregory Seumois, John Crawford, William Roberts, Stephen Schoenberger, Hilde Cheroutre, Christian Ottensmeier, Pandurangan Vijayanand. Single-cell transcriptomics and TCR clonality reveal a role for immunotherapy in pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB310.

Abstract LB075: Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models

Abstract Chimeric antigen receptor (CAR)-T cell therapy has been shown to effectively treat solid tumors in syngeneic murine models, but antigen loss and T cell trafficking into the tumors pose major obstacles to its long-term efficacy. Here we explored a combination therapy against advanced stages of colorectal and ovarian syngeneic murine tumor models using a tumor-specific small molecule (PAC-1) that triggers the engagement of the host immune system and synergizes with a Tn-specific adoptively transferred 237 CAR-T immunotherapy. PAC-1 has been shown to induce apoptosis in tumor cells with high expression of procaspase 3, via chelation of inhibitory zinc and release of active executioner caspase 3. PAC-1 also recently completed a phase I trial (NCT02355535) and was shown to be well-tolerated in late-stage cancer patients. RNA sequencing of CT26 colorectal cells stimulated with PAC-1 for 24 hours revealed that PAC-1 induced global changes at the transcriptome level, up-regulating genes involved in immune activation and apoptosis, and down-regulating genes involved in DNA repair, cell cycle regulation, and myc-driven proliferation. Validation of RNAseq data was performed using a combination of RT-qPCR, Western blot, flow cytometry, and co-culture studies. A combination of transcriptome and exome sequencing analyses in murine colorectal cell line CT26 revealed that the PAC-1-mediated down-regulation of DNA repair machinery led to somatic alterations in the genome and a higher mutational load in tumor cells, subsequently presented as neo-antigens by antigen-presenting cells. As a result, host CD8+ cytotoxic T cells were able to infiltrate tumors and reduce the tumor burden. Tn-dependent CARs have been used recently to demonstrate the proof of concept in the targeting of cancer-specific Tn-antigens such as the Tn-MUC1 antigen in humans, and an analogous Tn-OTS8 target in mice. In our murine ID8 ovarian tumor model, untreated mice with disseminated tumors have a median survival of 71 days. As a single agent administered intraperitoneally, PAC-1 extended the median survival to 90 days (p&amp;lt;0.0001), while 237 CAR-T treated mice have a median survival of 103 days (p&amp;lt;0.0001). A combination of the two treatment modalities (PAC-1 administered before 237 CAR-T) further extended the median survival to 145 days (p=0.048). Administering PAC-1 after 237 CAR-T also modestly extended the median survival to 132 days (p=0.06). Taken altogether, our results demonstrate that PAC-1 engages the immune system to treat cancer by inducing expression of pro-inflammatory cytokines in tumor cells allowing the recruitment of CD8+ effector T cells to the tumor microenvironment. Combination strategies that boost T cell trafficking shows great promise to enhance the anti-tumor efficacy and feasibility of adoptive CAR-T therapy against solid tumors. Citation Format: Diana R. Ranoa, Yifei Kang, Jenny Drnevich, Gloria Rendon, Christopher J. Fields, Keith Bailey, Edward J. Roy, Timothy M. Fan, David M. Kranz, Paul J. Hergenrother. Small molecule procaspase activating compound 1 (PAC-1) enhances CAR-T immunotherapy for solid tumors in syngeneic murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB075.