Cell-mediated Antitumor Immune Responses Research Articles

Cytosolic DNA composition is determined by genomic instability mechanism and regulates dendritic cell-mediated anti-tumor immunity.

Patients with colorectal cancers (CRCs) that have microsatellite instability (MSI) (MSI CRCs) face a better prognosis than those with the more common chromosomal instability (CIN) subtype (CIN CRCs) due to improved T cell-mediated anti-tumor immune responses. Previous investigations identified the cytosolic DNA (cyDNA) sensor STING as necessary for chemokine-mediated Tcell recruitment in MSI CRCs. Here, we find that cyDNA from MSI CRC cells is inherently more capable of inducing STING activation and improves cytotoxic Tcell activation by dendritic cells (DCs). Sequencing indicates that MSI cyDNA is enriched in microsatellites, which, upon DC uptake, induce anti-tumor immunity in a manner consistent with clinical MSI CRCs. DNA-damaging therapies also modulate cyDNA stimulation capacity, with radiation inducing larger cyDNA sizes and increased mitochondrial DNA content. Identifying highly stimulatory endogenous cyDNAs such as those in MSI CRCs will allow for optimized development of DNA-based STING agonist therapies to improve the responses of CIN CRCs with CIN to immunotherapies.

Enhanced Antitumor Efficacy of Oncolytic Vaccinia Virus Therapy Through Keratin-Mediated Delivery in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents an aggressive subtype characterized by high rates of recurrence and metastasis, necessitating the exploration of alternative treatment strategies. Oncolytic vaccinia virus (OVV) therapy has emerged as a promising approach, selectively infecting and destroying tumor cells. However, its efficacy is often hampered by inadequate viral distribution within the tumor microenvironment. Here, we investigate the potential of keratin (KTN) as a carrier for OVV delivery to enhance viral distribution and antitumor efficacy. In vitro assays revealed that KTN significantly improves OVV stability, leading to increased tumor cell apoptosis and necrosis. Furthermore, KTN effectively inhibits cancer cell migration by suppressing the epithelial-mesenchymal transition (EMT) process and downregulating metastasis-related proteins. These findings are corroborated in a syngeneic TNBC mouse model, where KTN-mediated OVV delivery enhances cytotoxic T cell-mediated antitumor immune responses without compromising the anti-angiogenic effects of the virus. Notably, KTN alone exhibits antitumor effects by suppressing tumor growth and metastasis, underscoring its potential as a standalone therapeutic agent. In conclusion, our study underscores the promise of KTN-mediated OVV delivery as a promising therapeutic strategy for TNBC. By improving viral distribution, suppressing EMT, and enhancing antitumor immunity, this approach holds significant potential for enhancing patient outcomes in TNBC treatment. Further investigation is warranted to explore the broader utility of KTN in various cancer therapy approaches.

Synergizing autophagic cell death and oxaliplatin-induced immunogenic death by a self-delivery micelle for enhanced tumor immunotherapy

Chemotherapy has become an emerging strategy to activate cytotoxic T cell responses by inducing immunogenic cell death (ICD), but the level of antitumor immunity induced by chemotherapeutic agents, such as oxaliplatin (OXA), is limited due to inadequate tumor antigen presentation and T cell activation. Inducing autophagic cell death (ACD) promotes the release of tumor antigen and the recruitment of dendritic cells, therefore strengthening antitumor immune responses. Here we simultaneously activate ICD and ACD with tumor targeting micelle to achieve enhanced antitumor chemo-immunotherapy. A self-delivery micelle is formulated by conjugating OXA prodrug with tocopherol succinate (TOS) as a hydrophobic segment and further encapsulates autophagy activator SMER28 to afford TOPR/SMER28, which specifically targets αvβ3 on tumor cells with c(RGDfK). Upon cellular internalization, OXA is released from the prodrug in response to the high concentration of reduced glutathione (GSH) in tumor cells, triggering ICD and releasing associated molecular patterns (DAMPs) signaling molecules to stimulate immunity. Meanwhile, SMER28 over-activates autophagy to induce autophagic cell death, which further leads to the maturation of dendritic cells and ultimately activates anti-tumor immune response. In the 4T1 tumor-bearing mice, the combination of OXA and SMER28 effectively inhibits tumor growth and activates antitumor immune responses. The tumor targeted micelle releases OXA and SMER28 in an on-demand profile and strengthens tumor chemo-immunotherapy by synergizing ICD and ACD, providing an alternative for antitumor immunotherapy. Statement of significanceChemotherapy induces immunogenic cell death (ICD) to activate anti-tumor immunity. However, the efficacy is limited by low levels of antigen presentation and T cell activation. To strengthen the antitumor immune responses induced by ICD, we first combine autophagic cell death (ACD) with ICD by formulating a glutathione-responsive oxaliplatin prodrug micelle co-encapsulating the autophagy activator SMER28. The activated autophagic level by SMER28 enhances the release of antigen and the recruitment of APCs, and ultimately bolsters T cell-mediated antitumor immune responses. We provide a potential strategy to amplify antitumor immune effects by combining autophagy activation with chemotherapy.

The ubiquitin-proteasome system in the tumor immune microenvironment: a key force in combination therapy.

The ubiquitin-proteasome system (UPS) plays a crucial role in modulating the proliferation, activation, and normal functioning of immune cells through the regulation of protein degradation and function. By influencing the expression of immune checkpoint-associated proteins, the UPS modulates T cell-mediated anti-tumor immune responses and can potentially facilitate the immune escape of tumor cells. Additionally, the UPS contributes to the remodeling of the tumor immunosuppressive microenvironment (TIME) by regulating B cells, dendritic cells (DCs), macrophages, and Treg cells. Targeting the UPS in conjunction with immune checkpoint-associated proteins, and combining these with other therapeutic approaches, may significantly enhance the efficacy of combination therapies and pave the way for novel cancer treatment strategies. In this review, we first summarize the composition and alterations of the TIME, with a particular emphasis on the role of the UPS in TIME and its interactions with various immune cell types. Finally, we explore the potential of combining UPS-targeted therapies with immunotherapy to substantially improve the effectiveness of immunotherapy and enhance patient survival outcomes.

H3K27M diffuse midline glioma is homologous recombination defective and sensitized to radiotherapy and NK cell-mediated antitumor immunity by PARP inhibition.

Radiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune responses. H3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1 inhibition by AZD9574 with concurrent RT. H3K27M mutation caused an HRR defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG tumor cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574. The HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of best-in-class PARP inhibitors with RT in DMG patients. H3K27M DMG are HRR defective and selectively radiosensitized by PARP inhibitor.PARP inhibitor with RT enhances NKG2D ligand expression and NK cell-mediated lysis.NK cells are required for the therapeutic efficacy of PARP inhibitor and RT. Radiotherapy is the cornerstone of H3K27M-mutant diffuse midline glioma treatment, but almost all patients succumb to tumor recurrence with poor overall survival, underscoring the need for RT-based precision combination therapy. Here, we reveal HRR deficiency as an H3K27M-mediated vulnerability and identify a novel mechanism linking impaired RT-induced histone H1 polyubiquitination and the subsequent RNF168/BRCA1/RAD51 recruitment in H3K27M DMG. This model is supported by selective radiosensitization of H3K27M DMG by PARP inhibitor. Notably, the combination treatment results in NKG2D ligand expression that confers susceptibility to NK cell killing in H3K27M DMG. We also show that the novel brain penetrant, PARP1-selective inhibitor AZD9574 compares favorably to olaparib when combined with RT, prolonging survival in a syngeneic orthotopic model of H3K27M DMG. This study highlights the ability of PARP1 inhibition to radiosensitize and induce an NK cell-mediated antitumor immunity in H3K27M DMG and supports future clinical investigation.

Picrasidine S Induces cGAS-Mediated Cellular Immune Response as a Novel Vaccine Adjuvant.

New adjuvants that trigger cellular immune responses are urgently needed for the effective development of cancer and virus vaccines. Motivated by recent discoveries that show activation of type I interferon (IFN-I) signaling boosts T cell immunity, this study proposes that targeting this pathway can be a strategic approach to identify novel vaccine adjuvants. Consequently, a comprehensive chemical screening of 6,800 small molecules is performed, which results in the discovery of the natural compound picrasidine S (PS) as an IFN-I inducer. Further analysis reveals that PS acts as a powerful adjuvant, significantly enhancing both humoral and cellular immune responses. At the molecular level, PS initiates the activation of the cGAS-IFN-I pathway, leading to an enhanced T cell response. PS vaccination notably increases the population of CD8+ central memory (TCM)-like cells and boosts the CD8+ T cell-mediated anti-tumor immune response. Thus, this study identifies PS as a promising candidate for developing vaccine adjuvants in cancer prevention.

Free Radicals Generated in Perfluorocarbon-Water (Liquid-Liquid) Interfacial Contact Electrification and Their Application in Cancer Therapy.

Electron transfer during solid-liquid contact electrification has been demonstrated to produce reactive oxygen species (ROS) such as hydroxyl radicals (•OH) and superoxide anion radicals (•O2-). Here, we show that such a process also occurs in liquid-liquid contact electrification. By preparing perfluorocarbon nanoemulsions to construct a perfluorocarbon-water "liquid-liquid" interface, we confirmed that electrons were transferred from water to perfluorocarbon in ultrasonication-induced high-frequency liquid-liquid contact to produce •OH and •O2-. The produced ROS could be applied to ablate tumors by triggering large-scale immunogenic cell death in tumor cells, promoting dendritic cell maturation and macrophage polarization, ultimately activating T cell-mediated antitumor immune response. Importantly, the raw material for producing •OH is water, so the tumor therapy is not limited by the endogenous substances (O2, H2O2, etc.) in the tumor microenvironment. This work provides new perspectives for elucidating the mechanism of generation of free radicals in liquid-liquid contact and provides an excellent tumor therapeutic modality.

CCAAT enhancer binding protein delta activates vesicle associated membrane protein 3 transcription to enhance chemoresistance and extracellular PD-L1 expression in triple-negative breast cancer

BackgroundChemoresistance and immunosuppression are two major obstacles in the current anti-cancer treatments. This study investigates the involvements of a CCAAT enhancer binding protein delta (CEBPD)/vesicle associated membrane protein 3 (VAMP3) axis in paclitaxel (PTX) resistance and immune evasion in triple-negative breast cancer (TNBC).MethodsPTX resistance-related genes were screened by bioinformatics. CEBPD and VAMP3 expression in clinical TNBC samples was examined by immunohistochemistry. Three PTX-resistant TNBC cell lines (MDA-MB-231/PTX, MDA-MB-468/PTX and MDA-MB-453/PTX) were generated, and their drug resistance was analyzed. Autophagy of cells was analyzed by immunofluorescence staining. Interaction between CEBPD and VAMP3 promoter was identified by immunoprecipitation and luciferase assays. The extracellular expression of programmed cell death-ligand 1 (PD-L1) in TNBC cells was detected. Extracellular vesicles (EVs) from TNBC cells were isolated to examine their effects on CD8+ T cell exhaustion.ResultsCEBPD and VAMP3 were upregulated in chemo-resistant tissue samples and in PTX-resistant TNBC cells. The CEBPD downregulation enhanced PTX sensitivity of cells. However, further upregulation of VAMP3 in cells restored PTX resistance, which was likely due to the activation of autophagy, as the autophagy antagonist chloroquine enhanced PTX sensitivity of cells. CEBPD was found to bind to the VAMP3 promoter to activate its transcription. The CEBPD/VAMP3 axis also increased the PD-L1 expression in the conditioned medium of TNBC cells. The TNBC cell-derived EVs increased the exhaustion of co-cultured CD8+ T cells.ConclusionThis study provides novel evidence that CEBPD plays a key role in enhancing PTX resistance in TNBC cells across various subtypes through VAMP3-mediated autophagy activation. Additionally, the CEBPD/VAMP3 axis also increases extracellular PD-L1 level, delivered by cancer cell-derived EVs, to suppress CD8+ T cell-mediated anti-tumor immune response. These significant observations may provide new insights into the treatment of TNBC, suggesting CEBPD and VAMP3 as promising targets to overcome treatment resistance.

Abstract LB081: Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma

Abstract Telomerase (TERT) is an enzyme critical for carcinogenesis expressed in about 90% of human cancers, including aggressive diffuse large B-cell lymphoma (DLBCL). Targeting TERT proven difficult due to on-target toxicities to non-malignant hematopoietic stem cells and potentially to activated immune effector cells. To overcome these challenges, we developed novel TLR9+ cell-selective CpG-oligodeoxynucleotides (ODNs) to deliver telomere-damaging nucleoside analogue, 6-thio-2’-deoxy-guanosine (6tdG), into target B cell lymphoma cells in vitro and in vivo. Human and mouse TLR9+/TERT+ lymphoma cells rapidly internalized CpG(6tdG)-oligonucleotides through active endocytosis. 6tdG molecules were released from the 3’ end of the oligonucleotide into cytosol and cell nucleus. TERT-mediated 6tdG incorporation into telomeres, led to DNA damage and lymphoma cell death. In vitro, CpG(6tdG)-oligonucleotides showed potency in targeting variety of human DLBCL cells in nanomolar range without detectable toxicity to non-malignant TERT— cells or human TERT+ hematopoietic stem cells and activated T cells. Importantly, the repeated systemic administration of CpG(6tdG)-oligonucleotides was well-tolerated by humanized hCD34/NOG mice, without detectable decrease in the percentage of hematopoietic stem cells, myeloid cells or T cells although the numbers of B cells were partly reduced as expected. Intravenously injected CpG(6tdG)-oligonucleotides, but not 6tdG nucleoside, had potent and dose-dependent antitumor effects against xenotransplanted models of human GC- or ABC-subtypes of DLBCL, such as OCI-Ly18 or OCI-Ly3, respectively. Furthermore, we assessed CpG(6tdG)-oligonucleotide activity against aggressive, syngeneic TERT+ Eμ-myc/15A lymphoma in immunocompetent mice. CpG(6tdG)-oligonucleotide, but not the CpG-oligonucleotide alone, 6tdG alone or co-injection of thereof, was effective in triggering regression of established 15A tumors with long-term survival of the majority of mice. The effect of CpG(6tdG)-oligonucleotide correlated with the activation of tumor antigen-specific T cell-mediated antitumor immune responses. In contrast, when tested in immunodeficient mice, CpG(6tdG)-oligonucleotide induced significant but transient regression of 15A lymphoma. These results suggest that the oligonucleotide elicits two-pronged activity triggering direct lymphoma cell cytotoxicity and also immune-mediated antitumor effects. Overall, this cell-selective strategy could provide an effective and safer strategy against aggressive TERT+ DLBCL cells with potential to engage and support T cell-based antitumor immunity. Citation Format: Chunsong Yu, Yong Liang, Piotr Swiderski, Larry Kwak, Marcin Kortylewski. Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma [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 LB081.

Abstract 5629: MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas

Abstract Background: Immune escape remains a challenge in the treatment of hepatocellular carcinoma (HCC), one of the most common and deadly cancers in the world. The transcription factor, myocyte enhancer factor 2D (MEF2D), is important in the regulation of differentiation and adaptive responses in many cancers, such that we tested its role in HCC. Methods: We knocked down MEF2D in HCC cell lines and analyzed HCC tissues and cell lines by RNA sequencing, Western blot and immunohistochemistry. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation assay. Chromatin immunoprecipitation was used to analyze the regulation of CD70 transcription by MEF2D. H22 cells, with MEF2D knockout or without (controls), were injected into the livers of syngeneic BALB/c mice. Flow cytometry was used to analyze the function of T cells in tumors, spleens, and lymph nodes. Results: Through database searches, we noted that MEF2D and CD70 were upregulated in HCC and associated with shorter patient survival times. Compared to WT tumors, injection of MEF2D-knockdown HCC cells into immunocompetent mice led to smaller tumors with increased T cell infiltration, activation, and impaired T-regulatory (Treg) cell suppressive function. Mechanistically, we found that MEF2D binds to the promoter region of CD70 gene and activates its transcription. Moreover, acetylation of MEF2D enhances the binding of MEF2D to the CD70 promoter and further promotes its transcription. CD70 blocking antibody inhibited activation of the CD70-CD27 signaling axis in murine HCC tumors, leading to impaired immunosuppressive function of Tregs and enhanced T cell-mediated anti-tumor immune responses. Thus, regulation of the CD70-CD27 signaling axis by MEF2D affects the numbers and functions of Treg cells and thereby controls T cell infiltration. Conclusions: MEF2D in HCC cells increases the expression of CD70 and blocks T cell-mediated anti-tumor immunity via the CD70-CD27 signaling axis. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC. Citation Format: Fanhua Kong, Liqing Wang, Qifa Ye, Yan Xiong, Wayne W. Hancock. MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas [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 5629.

Correlation between PD-L1 and Ki-67 Expression at various T-stage Clear Cell Renal Cell Carcinomas

Renal cell carcinoma (RCC) is a malignant neoplasm originating from renal epithelium, with the clear cell renal cell carcinoma (ccRCC)being the most common type (80%) and the most common cause of death among other types of kidney cancer. Pathological stage is an important parameter that affects ccRCC survival, followed by nuclear grade. Pathological staging of RCC according to the AJCC (American Joint Committee on Cancer) TNM system 8th edition is based on local extension of the main tumor (T), involvement of lymph node (N), and metastasis (M). Ki-67 is a marker of proliferation used to assess tumor grade. High Ki-67 correlates with poor prognosis, advanced clinical and pathological features, thus Ki-67 can be used as a biomarker in the management of RCC.Ki-67 is routinely used to see the proliferation index in various cases of malignancy, but not in kidney malignancy. Programmed death ligand 1 (PD-L1) acts as a negative regulator of T cell-mediated anti-tumor immune responses. PD-L1 is expressed on T cells, B cells, macrophages, dendritic cells, endothelial cells and in various tumor cells including ccRCC. This study aims to determine the correlation between the expression of PD-L1 and Ki-67 in various T-stage clear cell renal cell carcinomas. Material and Method: This was an observational analytical study with cross-sectional approach toward 52 cases of ccRCC whose diagnosis was made histopathologically at the Anatomical Pathology Installation of Dr. Soetomo General Academic Hospital Surabaya from January 2014 to December 2020. Immunohistochemical stainingwas carried out using Ki-67 and PD-L1 antibodies, followed by an assessment using a scoring system. T-stage data were obtained from the patients’ medical recordswhich were then analyzed statistically with the Spearman test. Result: The study included 52 cases of ccRCC obtained from nephrectomy specimens at RSUD dr. Soetomo between 2014–2020. The age distribution of the subjects was 29–69 years and the mean and median age was 53 years. The ratio of male patients compared to female patients was 2.5:1. The majority was stage T2 (50%). Statistical test results showed no correlation between the expression of PD-L1 and Ki-67 in various T-stage clear cell renal cell carcinomas (p=0.965 and p=0.680). Conclusion: This study showed no correlation between the expression of PD-L1 and Ki-67 in various T-stage clear cell renal cell carcinomas. Nonetheless PD-L1 can be considered as an important biomarker with a poorer prognosis and aggressive clinicopathological findings in patients with RCC.

Interleukin-36gamma Mediates the In Vitro Activation of CD8+ T Cells from Patients Living with Chronic Human Immunodeficiency Virus-1 Infection.

Interleukin-36 (IL-36) signaling plays an important role in promoting CD8+ T cell-mediated antitumor immune responses. The role of IL-36 signaling in CD8+ T cells that are involved in host immune responses during human immunodeficiency virus-1 (HIV-1) infection has not been characterized. Sixty-one patients living with chronic HIV-1 infection and 23 controls were enrolled in this study. The levels of IL-36 cytokine family members were measured by enzyme-linked immunosorbent assay. Purified CD8+ T cells were stimulated with recombinant IL-36gamma (1 or 10 ng/mL). The expression of inhibitory receptors, the secretion of cytotoxic molecules and interferon-gamma, and the mRNA levels of apoptosis-related ligands were assessed to evaluate the effect of IL-36gamma on CD8+ T cell function in vitro. There were no significant differences in IL-36alpha, IL-36beta, or IL-36 receptor antagonist levels between patients living with chronic HIV-1 infection and controls. Plasma IL-36gamma levels were reduced in patients living with chronic HIV-1 infection. Perforin, granzyme B, and granulysin secretion, as well as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) mRNA expression, but not programmed death-1 (PD-1) or cytotoxic T lymphocyte-associated protein-4 (CTLA-4) expression was downregulated in CD8+ T cells from patients living with chronic HIV-1 infection. The addition of both 1 and 10 ng/mL IL-36gamma enhanced perforin, granzyme B, granulysin, and interferon-gamma secretion by CD8+ T cells without affecting PD-1/CTLA-4 or TRAIL/FasL mRNA expression in CD8+ T cells from patients living with chronic HIV-1 infection. The addition of 1 ng/mL IL-36gamma also promoted perforin and granzyme B secretion by HIV-1-specific CD8+ T cells from patients living with chronic HIV-1 infection. The reduced IL-36gamma levels in patients living with chronic HIV-1 infection might be insufficient for the activation of CD8+ T cells, leading to CD8+ T cell exhaustion.

Predictive Biomarkers of Immune Checkpoint Inhibitor-Based Mono- and Combination Therapies for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is among the most frequent and deadly human cancers worldwide. It has been shown that interaction between immune checkpoint receptors and ligands plays a crucial role in inhibition of T cell-mediated anti-tumor immune responses, thereby assisting tumor cells to evade the host immune surveillance. Therefore, several immune checkpoint inhibitors (ICIs) that selectively block immune checkpoint receptors or ligands have been developed as clinically effective and safe immunotherapeutic agents for treating HCC, including the inhibitors targeting cytotoxic T lymphocyte-associated antigen 4, programmed death 1, and programmed death ligand 1. In addition, various combinations of ICIs and other ICIs or tyrosine kinase inhibitors or vascular endothelial growth factor inhibitors have also emerged as clinically beneficial treatments for HCC. However, the overall response rates of ICI mono-therapy and combination therapy in HCC patients remain unsatisfied, highlighting the urgent need for discovering valuable predictive biomarkers to achieve personalized therapy. This review comprehensively summarizes the literature-based evidence validating a variety of biomarkers with predictive significance for treatment responses and outcomes in HCC patients receiving various ICI-based mono- and combination therapies.

Combined TIM-3 and PD-1 blockade restrains hepatocellular carcinoma development by facilitating CD4+ and CD8+ T cell-mediated antitumor immune responses.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) are beneficial to the resumption of anti-tumor immunity response and hold extreme potential as efficient therapies for certain malignancies. However, ICIs with a single target exhibit poor overall response rate in hepatocellular carcinoma (HCC) patients due to the complex pathological mechanisms of HCC. To investigate the effects of combined TIM-3 and PD-1 blockade on tumor development in an HCC mouse model, aiming to identify more effective immunotherapies and provide more treatment options for HCC patients. The levels of PD-1 and TIM-3 on CD4+ and CD8+ T cells from tumor tissues, ascites, and matched adjacent tissues from HCC patients were determined with flow cytometry. An HCC xenograft mouse model was established and treated with anti-TIM-3 monoclonal antibody (mAb) and/or anti-PD-1 mAb. Tumor growth in each group was measured. Hematoxylin and eosin staining and immunohistochemical staining were used to evaluate T cell infiltration in tumors. The percentage of CD4+ and CD8+ T cells in tissue samples from mice was tested with flow cytometry. The percentages of PD-1+CD8+, TIM-3+CD8+, and PD-1+TIM-3+ CD8+ T cells was accessed by flow cytometry. The levels of the cytokines including tumor necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6, and IL-10 in tumor tissues were gauged with enzyme-linked immunosorbent assay kits. We confirmed that PD-1 and TIM-3 expression was substantially upregulated in CD4+ and CD8+ T cells isolated from tumor tissues and ascites of HCC patients. TIM-3 mAb and PD-1 mAb treatment both reduced tumor volume and weight, while combined blockade had more substantial anti-tumor effects than individual treatment. Then we showed that combined therapy increased T cell infiltration into tumor tissues, and downregulated PD-1 and TIM-3 expression on CD8+ T cells in tumor tissues. Moreover, combined treatment facilitated the production of T cell effector cytokines TNF-α and IFN-γ, and reduced the production of immunosuppressive cytokines IL-10 and IL-6 in tumor tissues. Thus, we implicated that combined blockade could ameliorate T cell exhaustion in HCC mouse model. Combined TIM-3 and PD-1 blockade restrains HCC development by facilitating CD4+ and CD8+ T cell-mediated antitumor immune responses.

Abstract A169: Overcoming tumor-associated immune suppression and resistance to cancer immunotherapy using Hyal2-ADCC

Abstract Background: Multiple clinical studies have addressed the role of immunotherapies for solid cancers. While these studies have demonstrated promising results and improvements over existing therapies, less than half of patients with advanced disease show the clinical benefits of immunotherapy. Furthermore, once tumors start to produce metastases, it becomes one of the leading causes of cancer-related mortality. Hyal2-ADC is a novel conjugate of anti-Hyal2 monoclonal antibody and cytotoxic payload PNU. Hyaluronidase 2 (Hyal2), which is a membrane-bound enzyme expressed by both MDSCs and epithelial tumor cells, serves as a molecular target for the Hyal2-ADC. Importantly, our anti-Hyal2 antibodies recognize both human and mouse Hyal2. Methods: Expression of Hyal2 in human and mouse cancer tissues was assessed using immunohistochemistry and Western Blotting. The in vitro effects of Hyal2-ADC tumor cells and MDSCs were evaluated using MTT assay and immunofluorescence. A single-cell RNA sequencing was utilized for the transcriptomic analysis of Hyal2-expressing myeloid cells. The in vivo anti-tumor activity of Hyal2-ADC was evaluated using syngeneic mouse tumor models and human xenograft tumor models in immunodeficient mice. Mice with established bladder or prostate tumors were used in this study. Results: Administration of Hyal-ADC alone led to the inhibition of tumor growth in a dose-dependent manner. Importantly, the therapeutic effect of Hyal2-ADC was associated with remodeling the tumor microenvironment, reduction of immunosuppressive myeloid cells, and influx of immune T cells. A combination of Hyal2-ADC with anti-PD1 therapy resulted in complete tumor eradication in mice with syngeneic established tumors (initial volume <200 mm3) in 100% treated mice. The cured mice demonstrated long-term protection from tumor re-challenge. Furthermore, the combination therapy of mice with large, advanced established tumors (>200mm3) resulted in 80% tumor eradication. Conclusions: Hyal2-ADC is a first-in-class drug that selectively targets Hyal2-expressing cells. The modulation of the immunosuppressive tumor microenvironment and development of efficient immune memory observed in murine models suggests that Hyal2-ADC alone or in combination with anti-PD1 therapy could be utilized for the treatment of solid tumors associated with deregulated HA metabolism, including bladder, prostate, breast, brain, and other cancers. Mechanistically, the enzyme Hyal2 is involved in the degradation of tumor-associated hyaluronan (HA), one of the major components of the extracellular matrix in tumor stroma. Depletion of Hyal2-expressing cells leads to normalization of HA metabolism in the tumor microenvironment supporting generation of T cell-mediated anti-tumor immune response. Overall, this study introduces a novel concept to remodeling the tumor microenvironment, bypassing tumor-associated immune suppression, and overcoming resistance to cancer immunotherapy, thus representing a novel therapeutic strategy for patients. Citation Format: Sergei Kusmartsev, William Donelan, Wayne Brisbane, Brianna Nguyen, Meng-Lun Hsieh, Shiwu Li, Paul Dominguez-Gutierrez, Padraic O'Malley, Paul L Crispen. Overcoming tumor-associated immune suppression and resistance to cancer immunotherapy using Hyal2-ADCC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A169.

Abstract IA015: Leveraging mRNA to design a next-generation cell-based therapeutic cancer vaccine

Abstract Monocyte-derived dendritic cell (DC)-based cancer vaccines were shown to activate T cell-mediated antitumor immune responses. To ensure high tumor specificity and potency of the DC-vaccine, we leveraged mRNA for the delivery of tumor antigens and proteins that enhance the DCs’ T-cell stimulatory capacity. With regard to tumor antigens, we studied neoantigens. These originate from erroneous DNA in tumor cells, creating a message for novel and unique tumor antigens of which neoepitopes could be presented by the tumor cells to T cells. Identifying neoepitopes has become possible through advances in next-generation sequencing and bioinformatic prediction algorithms. We developed a synthetic DNA template (SDT) to manufacture in vitro transcribed neoepitope mRNA. A benefit of this platform is the high flexibility and speed to create a neoepitope mRNA library. We showed that mRNA produced with the SDT could be used to validate bioinformatically predicted neoepitopes and could potentially be used to produce a DC-vaccine. With regard to potency, we developed a novel mRNA-based adjuvant, building on the knowledge that interleukin-12 produced by DCs correlates with T-cell activation. The novel mRNA mix, referred to as TetraMix, consists of 4 mRNA molecules encoding (1) constitutively active toll-like receptor 4, (2) CD40 ligand, (3) interferon-gamma and (4) a decoy receptor for interleukin-10. DCs electroporated with TetraMix mRNA showed a viral response gene expression profile. The obtained DC-maturation state enabled activation of tumor killing T cells. In summary, we developed a personalized cancer vaccine consisting of mRNA-engineered DCs, which is ready for evaluation in a clinical setting.</aaff> Citation Format: Karine Breckpot. Leveraging mRNA to design a next-generation cell-based therapeutic cancer vaccine [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr IA015.

Targeting myeloid checkpoint Siglec-10 reactivates antitumor immunity and improves anti-programmed cell death 1 efficacy in gastric cancer

ObjectiveImmunotherapy has not yielded satisfactory therapeutic responses in gastric cancer (GC). However, targeting myeloid checkpoints holds promise for expanding the potential of immunotherapy. This study aims to evaluate the critical...

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

Photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes to promote PD-L1 multivalent binding for effective immune checkpoint blockade therapy

Immune checkpoint blockade (ICB) therapy targeting PD-L1 via monoclonal antibody (mAb) has shown extensive clinical benefits in the diverse types of advanced malignancies. However, most patients are completely refractory to ICB therapy owing to the PD-L1 recycling mechanism. Herein, we propose photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes (immune checkpoint blockade liposomes; ICB-LPs) to promote PD-L1 multivalent binding for inducing lysosomal degradation of PD-L1 in tumor cells. The ICB-LPs are prepared by formulation of DC8,9PC with photo-polymerized diacetylenic moiety, 1,2-dipalmitoylphosphatidylcholine (DPPC) and anti-PD-L1 peptide (D-form NYSKPTDRQYHF)-conjugated DSPE-PEG2k (anti-PD-L1-DSPE-PEG2k) in a molar ratio of 45:45:10, followed by cross-linking of liposomal bilayer upon UV irradiation. The 10 mol% anti-PD-L1-DSPE-PEG2k incorporated ICB-LPs have a nano-sized lipid bilayer structure with an average diameter of 137.7 ± 1.04 nm, showing a high stability in serum condition. Importantly, the ICB-LPs efficiently promote the multivalent binding with PD-L1 on the tumor cell membrane, which are endocytosed with aim to deliver PD-L1 to the lysosomes, wherein the durable PD-L1 degradation is observed for 72 h, in contrast to anti PD-L1 mAbs showing the rapid PD-L1 recycling within 9 h. The in vitro co-culture experiments with CD8+ T cells show that ICB-LPs effectively enhance the T cell-mediated antitumor immune responses against tumor cells by blocking the PD-L1/PD-1 axis. When ICB-LPs are intravenously injected into colon tumor-bearing mice, they efficiently accumulate within the targeted tumor tissues via both passive and active tumor targeting, inducing a potent T cell-mediated antitumor immune response by effective and durable PD-L1 degradation. Collectively, this study demonstrates the superior antitumor efficacy of crosslinked and anti-PD-L1 peptide incorporated liposome formulation that promotes PD-L1 multivalent binding for trafficking of PD-L1 toward the lysosomes instead of the recycling endosomes.

Paeoniflorin inhibits hepatocellular carcinoma growth by reducing PD-L1 expression

Abnormal expression of programmed death-ligand 1 (PD-L1) on cancer cells contributes to immune escape in hepatocellular carcinoma (HCC). Paeoniflorin has been shown to inhibit the growth of HCC; however, whether its inhibitory effect involves reducing PD-L1 expression on HCC cells remains unknown. We investigated the antitumor effects of paeoniflorin and its potential regulatory mechanisms in HCC. The effects of paeoniflorin on tumor growth and tumor immunity were determined in H22-xenografted mice and DEN-induced HCC rats. Small interfering RNA against suppressor of cytokine signaling 3 (SOCS3) was transfected into HepG2 cells to verify the effect of paeoniflorin on the SOCS3/signal transducer and activator of transcription 3 (STAT3)/PD-L1signaling pathway. The levels of SOCS3/STAT3/PD-L1 signaling pathway-related mRNAs and proteins were determined by real time-polymerase chain reaction and western blotting, respectively. Interleukin-2 (IL-2), interferon-γ (IFN-γ), granzyme B (GrB), and perforin 1 (PRF1) levels were detected in an H22 and mouse T cell co-culture system. Paeoniflorin can trigger T cell-mediated anti-tumor immune responses by increasing CD8+ T cell counts in tumor tissues, thereby inhibiting tumor growth. Moreover, paeoniflorin increased IL-2, IFN-γ, GrB, and PRF1 levels in the co-culture system. PD-L1 expression was suppressed by paeoniflorin, and this effect was mediated by the SOCS3/STAT3 signaling pathway. Paeoniflorin might thus act via enhancing SOCS3 to inhibit STAT3/PD-L1 signaling and subsequently restore T cell sensitivity to kill tumor cells. Our findings provide novel insights into the anticancer effects of paeoniflorin.