Activation Of Antitumor Immunity Research Articles

Boron Neutron Capture Therapy-Derived Extracellular Vesicles via DNA Accumulation Boost Antitumor Dendritic Cell Vaccine Efficacy.

Radiated tumor cell-derived extracellular vesicles (RT-EVs) encapsulate abundant DNA fragments from irradiated tumor cells, in addition to acting as integrators of multiple tumor antigens. Accumulating evidence indicates these DNA fragments from damaged cells are involved in downstream immune responses, but most of them are degraded in cells before incorporation into derived RT-EVs, thus the low abundance of DNA fragments limits immune responses of RT-EVs. Here, this study found that different radiations affected fates of DNA fragments in RT-EVs. Boron neutron capture therapy (BNCT) induced DNA accumulation in RT-EVs (BEVs) by causing more DNA breaks and DNA oxidation resisting nuclease degradation. This is attributed to the high-linear energy transfer (LET) properties of alpha particles from the neutron capture reaction of 10B. When being internalized by dendritic cells (DCs), BEVs activated the DNA sensing pathway, resulting in functional enhancements including antigen presentation, migration capacity, and cytokine secretion. After vaccination of the BEVs-educated DCs (BEV@BMDCs), the effector T cells significantly expanded and infiltrated into tumors, suggesting robust anti-tumor immune activation. BEV@BMDCs not only effectively inhibited the primary tumor growth and metastasis formation but also elicited long-term immune memory. In conclusion, a successful DC vaccine is provided as a promising candidate for tumor vaccine.

Novel Carbon Quantum Dots Precisely Trigger Ferroptosis in Cancer Cells through Antioxidant Inhibition Synergistic Nanocatalytic Activity.

High levels of glutathione (GSH) are an important characteristic of malignant tumors and a significant cause of ineffective treatment and multidrug resistance. Although reactive oxygen species (ROS) therapy has been shown to induce tumor cell death, the strong clearance effect of GSH on ROS significantly reduces its therapeutic efficacy. Therefore, there is a need to develop new strategies for targeting GSH. In this study, novel carbon quantum dots derived from gentamycin (GM-CQDs) were designed and synthesized. On the basis of the results obtained, GM-CQDs contain sp2 and sp3 carbon atoms as well as nitrogen oxygen groups, which decrease the intracellular levels of GSH by downregulating SLC7A11, thereby disrupting redox balance, mediating lipid peroxidation, and inducing ferroptosis. Transcriptome analysis demonstrated that GM-CQDs downregulated the expression of molecules related to GSH metabolism while significantly increasing the expression of molecules related to ferroptosis. The in vivo results showed that the GM-CQDs exhibited excellent antitumor activity and immune activation ability. Furthermore, because of their ideal biological safety, GM-CQDs are highly promising for application as drugs targeting GSH in the treatment of malignant tumors.

89Zr-immunoPET-guided selection of a CD33xIL15 fusion protein optimized for antitumor immune cell activation and in vivo tumour retention in acute myeloid leukaemia.

Immune cells are capable of eliminating leukemic cells, as evidenced by outcomes in hematopoietic cell transplantation (HCT). However, patients who fail induction therapy will not benefit from HCT due to their minimal residual disease (MRD) status. Thus, we aimed to develop an immunomodulatory agent to reduce MRD by activating immune effector cells in the presence of leukaemia cells via a novel fusion protein that chimerises two clinically tolerated biologics: a CD33 antibody and the IL15Ra/IL15 complex (CD33xIL15). We generated a set of CD33xIL15 fusion protein constructs with varying configurations and identified those with the best in vitro AML-binding, T cell activation, and NK cell potentiation. Using 89Zr-immunoPET imaging we then evaluated the biodistribution and in vivo tumour retention of the most favourable CD33xIL15 constructs in an AML xenograft model. Ex vivo biodistribution studies were used to confirm the pharmacokinetics of the constructs. Two of the generated fusion proteins, CD33xIL15 (N72D) and CD33xIL15wt, demonstrated optimal in vitro behaviour and were further evaluated in vivo. These studies revealed that the CD33xIL15wt candidate was capable of being retained in the tumour for as long as its parental CD33 antibody, Lintuzumab (13.9 ± 3.1%ID/g vs 18.6 ± 1.1%ID/g at 120 h). This work demonstrates that CD33xIL15 fusion proteins are capable of targeting leukemic cells and stimulating local T cells in vitro and of concentrating in the tumour in AML xenografts. It also highlights the importance of 89Zr-immunoPET to guide the development and selection of tumour-targeted antibody-cytokine fusion proteins.

In Situ Aggregated Nanomanganese Enhances Radiation-Induced Antitumor Immunity.

Radiosensitizers play a pivotal role in enhancing radiotherapy (RT). One of the challenges in RT is the limited accumulation of nanoradiosensitizers and the difficulty in activating antitumor immunity. Herein, a smart strategy was used to achieve in situ aggregation of nanomanganese adjuvants (MnAuNP-C&B) to enhance RT-induced antitumor immunity. The aggregated MnAuNP-C&B system overcomes the shortcomings of small-sized nanoparticles that easily flow back into blood vessels and diffuse into surrounding tissues, and it also prolongs the retention time of nanomanganese within cancer cells and tumors. The MnAuNP-C&B system significantly enhances the radiosensitization effect in RT. Additionally, the pH-responsive disassembly of MnAuNP-C&B triggers the release of Mn2+, further promoting RT-induced activation of the STING pathway and eliciting robust antitumor immunity. Overall, our study presents a smart strategy wherein in situ aggregation of nanomanganese effectively inhibits tumor growth through radiosensitization and the activation of antitumor immunity.

DIPG-32. NEOANTIGEN HEAT SHOCK PROTEIN VACCINE, RHSC-DIPGVAX, IS ASSOCIATED WITH INCREASED VACCINE-INDUCED B CELLS AND BCR/TCR REPERTOIRE DIVERSITY IN PEDIATRIC PATIENTS WITH DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Diffuse midline gliomas (DMG/DIPG) remain a devastating class of tumors with limited treatment options. Cancer vaccines educate the immune system to tumor neoantigens to facilitate immune-mediated tumor destruction. DMGs have high-frequency, co-occurring neoantigens and are therefore a relevant target for activation of anti-tumor immunity via vaccine. To this end, we have initiated a clinical trial (NCT04943848) to determine if a neoantigen peptide-based vaccine with recombinant human heat-shock constitutive protein 70 and QS-21 adjuvant can promote anti-tumor immunity against DMG/DIPG. NCT04943848 is ongoing and now enrolling subsequent arms with rHSC-DIPGVax plus checkpoint blockade. METHODS Whole blood was collected before the first vaccine cycle (C1D1). Vaccine doses were administered every 2 weeks and blood was collected prior to the third or fifth vaccine cycle (C3D1 & C5D1). Peripheral blood mononuclear cells (PBMCs) were isolated and stored at -160°C. PBMCs from each patient, pre- and post-vaccine, were stained with human T-cell, myeloid, and B-cell antibody panels before spectral flow cytometry, single-cell RNA sequencing, and genomic DNA isolation for T- and B-cell receptor (TCR/BCR) sequencing. Spectral flow cytometry data was analyzed via FlowJo v10. TCR/BCR sequencing was processed via MiXCR and Immunarch. RESULTS Following vaccination, there were higher percentages of two mature CD20+CD19+IgM+ B cell populations, including a 4-1BBL+CD86+ population previously shown to induce robust CD8+ T-cell activation in adults with glioblastoma. Interestingly, given the recent discovery of TIM3’s importance in DIPG, we also identified TIM3-expressing CD4+ and CD8+ T cell populations. Finally, TCR/BCR sequencing demonstrated increased receptor repertoire diversity after vaccination, which is indicative of the vaccine shaping adaptive immunity. CONCLUSIONS Peripheral immune populations and TCR/BCR sequences change during vaccination with rHSC-DIPGVax. Ongoing analyses will determine the specificity of the patient immune response to vaccine antigens and correlate changes in immune populations to clinical course.

A combination of a TLR7/8 agonist and an epigenetic inhibitor suppresses triple-negative breast cancer through triggering anti-tumor immune

BackgroundCombination therapy involving immune checkpoint blockade (ICB) and other drugs is a potential strategy for converting immune-cold tumors into immune-hot tumors to benefit from immunotherapy. To achieve drug synergy, we developed a homologous cancer cell membrane vesicle (CM)-coated metal-organic framework (MOF) nanodelivery platform for the codelivery of a TLR7/8 agonist with an epigenetic inhibitor.MethodsA novel biomimetic codelivery system (MCM@UN) was constructed by MOF nanoparticles UiO-66 loading with a bromodomain-containing protein 4 (BRD4) inhibitor and then coated with the membrane vesicles of homologous cancer cells that embedding the 18 C lipid tail of 3M-052 (M). The antitumor immune ability and tumor suppressive effect of MCM@UN were evaluated in a mouse model of triple-negative breast cancer (TNBC) and in vitro. The tumor immune microenvironment was analyzed by multicolor immunofluorescence staining.ResultsIn vitro and in vivo data showed that MCM@UN specifically targeted to TNBC cells and was superior to the free drug in terms of tumor growth inhibition and antitumor immune activity. In terms of mechanism, MCM@UN blocked BRD4 and PD-L1 to prompt dying tumor cells to disintegrate and expose tumor antigens. The disintegrated tumor cells released damage-associated molecular patterns (DAMPs), recruited dendritic cells (DCs) to efficiently activate CD8+ T cells to mediate effective and long-lasting antitumor immunity. In addition, TLR7/8 agonist on MCM@UN enhanced lymphocytes infiltration and immunogenic cell death and decreased regulatory T-cells (Tregs). On clinical specimens, we found that mature DCs infiltrating tumor tissues of TNBC patients were negatively correlated with the expression of BRD4, which was consistent with the result in animal model.ConclusionMCM@UN specifically targeted to TNBC cells and remodeled tumor immune microenvironment to inhibit malignant behaviors of TNBC.

ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ Tcell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

Hyperosmotic cold shock mouse melanoma cells encapsulated with doxorubicin for targeted treatment of melanoma.

The primary treatment strategies for melanoma include surgical excision, chemotherapy, and radiotherapy. However, the efficacy of these treatments is often limited by drug resistance, recurrence, and severe side effects. Therefore, we aimed to develop a targeted drug delivery system capable of selectively locating tumor sites to minimize systemic toxicity and enhance therapeutic efficacy. This cell drug delivery system can also deliver chemotherapeutic drugs to the tumor microenvironment. We treated B16F10 cells with hyperosmotic cold shock (HCS) to obtain and characterize HCS cells. We then investigated the anti-tumor effects and immune activation capabilities of these cells and explored their potential as a targeted drug delivery system. HCS cells not only maintained an intact cellular structure and tumor antigens but also exhibited high expression of the homologous melanoma-associated antigen glycoprotein 100. These cells demonstrated an exceptional capacity for loading and releasing doxorubicin, which has chemotherapeutic anti-tumor effects. HCS cells can precisely target the tumor microenvironment to minimize systemic toxicity, inducing an immune response by activating CD3+ and CD4+ T cells. HCS cells are non-carcinogenic, with both cellular and tumor antigens intact; thus, they are suitable drug delivery carriers. Our findings highlight the potential of HCS cells for carrying doxorubicin because of their high drug-loading efficiency, effective tumor-targeting and anti-tumor effects. Therefore, our results will facilitate the development of melanoma treatments that have higher efficacy than those in the literature.

LncRNAs in Immune and Stromal Cells Remodel Phenotype of Cancer Cell and Tumor Microenvironment.

Emerging studies suggest that long non-coding RNAs (lncRNAs) participate in the mutual regulation of cells in tumor microenvironment, thereby affecting the anti-tumor immune activity of immune cells. Additionally, the intracellular pathways mediated by lncRNAs can affect the expression of immune checkpoints or change the cell functions, including cytokines secretion, of immune and stromal cells in tumor microenvironment, which further influences cancer patients' prognosis and treatment response. With the in-depth research, lncRNAs have shown great potency as a new immunotherapy target and predict immunotherapy response. The research on lncRNAs provides us with a new insight into developing new immunotherapy drugs and predicting the outcome of immunotherapy. With development of RNA sequencing technology, amounts of lncRNAs were found to be dysregulated in immune and stromal cells rather than tumor cells. These lncRNAs function through ceRNA network or regulating transcript factor activity, thus leading abnormal differentiation and activation of immune and stromal cells. Here, we review the function of lncRNAs in the immune microenvironment and focus on the alteration of lncRNAs in immune and stromal cells, and discuss how these alterations affect tumor growth, metastasis and treatment response.

Functional remodeling of intraperitoneal macrophages by oncolytic adenovirus restores anti-tumor immunity for peritoneal metastasis of gastric cancer

Intraperitoneal tumor-associated macrophages (TAMs) are involved in evading anti-tumor immunity and promoting the peritoneal metastasis (PM) of gastric cancer (GC). Oncolytic viruses are known to induce the activation of host anti-tumor immunity in addition to tumor lysis. This study investigated whether a wild-type p53-loading telomerase-specific oncolytic adenovirus (OBP-702) could elicit the remodeling of intraperitoneal macrophages and enhance the efficacy of immune therapy. Increased numbers of CD163 TAMs and few CD8+ lymphocytes were immunohistochemically observed in clinical samples with PM, which suggested that TAMs were associated with the suppression of anti-tumor immunity. OBP-702 induced immunogenic cell death and upregulated PD-L1 expression in human and murine GC cell lines. Intraperitoneal administration of OBP-702 increased recruitment of CD8+ lymphocytes into the PM via the functional remodeling of intraperitoneal macrophages from TAM towards a pro-inflammatory phenotype, resulting in significantly suppressed tumor growth for the in vivo model. Furthermore, the combination of intraperitoneal OBP-702 with anti-PD-1 Antibody enhanced anti-tumor immunity and prolonged the survival of mice bearing PM. Intraperitoneal immunotherapy using OBP-702 restores anti-tumor immunity via the remodeling of intraperitoneal macrophages in addition to direct tumor lysis and cooperates with immune checkpoint inhibitors to suppress PM in GC.

Nano-inducer of ferroptosis for targeted chemotherapy of human triple negative breast carcinoma

Triple negative breast carcinoma (TNBC) accounts for 15–20 % of all incident breast cancers (BC) and is known to be highly invasive, has fewer treatment options, and tends to have a worse prognosis. However, due to its biological heterogeneity and diverse clinical and epidemiological behaviors, TNBC lacks a tumor-specific targeted therapy. In the present work we have developed a TNBC-specific targeted nano-delivery agent comprising of a cRGD labeled magneto-liposome (T-LMD) co-encapsulated with oleic acid coated iron oxide nanoparticles (MN-OA) and doxorubicin (Dox) in the liposome bilayer and core, respectively. T-LMD was found to show enhanced uptake and induction of ferroptotic cell death in MDA-MB-231, a TNBC model cell line. Additionally, T-LMD induced ferroptosis was found to be accompanied by release of HMGB1, an immunogenic cell death marker, suggesting its immunogenicity for augmenting the activation of anti-tumor immunity in TNBC. The strategic placement of IONPs in the liposome bilayer of T-LMD facilitates the sensitization of MDA-MB-231 cells to undergo ferroptosis; predominantly via the activation of the iron/lipid metabolism pathway, as validated by use of small molecule ferroptosis inhibitor (ferrostatin-1) and iron chelator (deferoxamine). Activation of ferroptotic cell death was also corroborated by ferroptosis specific-ultrastructural alterations in the shape/size of cellular mitochondria and cell ballooning as observed by transmission electron microscopy and bright field imaging, respectively. Thus, our ferroptosis nano-inducer (T-LMD) can efficiently kill TNBC cells via enhanced LPO and ROS generation leading to membrane damage and consequent release of LDH and HMGB1, induce mitochondrial alterations and enhanced DNA double strand breaks. Altogether, our results suggest significant implications of T-LMD for treatment of TNBC.

High Manganese Content of Lipid NanoMn (LNM) by Microfluidic Technology for Enhancing Anti-Tumor Immunity.

Immunotherapy is a clinically effective method for treating tumors. Manganese can activate the cGAS-STING signaling pathway and induce an anti-tumor immune response. However, its efficacy is hindered by non-specific distribution and low uptake rates. In this study, we employed microfluidic technology to design and develop an innovative preparation process, resulting in the creation of a novel manganese lipid nanoparticle (LNM). The lipid manganese nanoparticle produced in this process boasts a high manganese payload, excellent stability, the capacity for large-scale production, and high batch repeatability. LNM has effectively demonstrated the ability to activate the cGAS-STING signaling pathway, induce the production of pro-inflammatory cytokines, and inhibit tumor development. Notably, LNM does not require combination chemotherapy drugs or other immune activators. Therefore, LNM presents a safe, straightforward, and efficient strategy for anti-tumor immune activation, with the potential for scalable production.

Abstract LB426: Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway

Abstract IDO1 is an immune checkpoint regulator and mediator of tumor immune evasion (1). The cellular and molecular mechanisms of IDO1-mediated immune suppression are not fully understood. We describe here that GPR35, the putative receptor for kynurenic acid (KYNA), a metabolite of IDO1-mediated tryptophan catabolism, couples to Hippo-YAP pathway. GPR35 activation, either by overexpression or by treatment with tryptophan metabolites resulted in Hippo inactivation and YAP-directed transcription via TEAD family of transcription factors. Recently, YAP is found to be essential for tumor immune escape. Yap1 deficiency in T-lineage-specific knockout mice, or by dosing a YAP inhibitor, impairs Treg formation and function, leading to tumor growth inhibition (2). YAP also plays an inhibitory role in CD8 T cell function, especially in activated CTL usually found in the TME [3,4]. Through medicinal chemistry design and a structure-vs-activity relationship study effort, we discovered a GPR35-selective inhibitor, here termed TMER1i. TMER1i potently blocks GPR35 in Hippo suppression and YAP activation. We showed that GPR35 is expressed in the T-cell lineage of the immune cells including naïve CD4 T cells, Treg and CD8 T cells. Treatment of purified human naïve CD4 T cells with anti-CD3/anti-CD28 under an IDO1+ TME-like culture condition in vitro induced a robust T cell activation and differentiation toward Treg phenotype, as shown previously using mouse naïve CD4 T cells by others (5). Importantly, TMER1i efficiently blocked the Treg differentiation under these conditions. When dosed in human-PBMC reconstituted NCG mice bearing HT29 tumors, TMER1i dose-dependently increased the infiltration of human CD8 T cells within tumors in a statistically significant manner, and a trending lower Treg cell infiltration, accompanied by a tumor growth inhibition. qPCR analysis of the HT29 tumors grown in the PBMC-NCG mice indicated that IDO1 was highly expressed in the tumors. These results indicated that GPR35 selective inhibitor TMER1i enhanced an anti-tumor immune activity in an IDO1+ tumor microenvironment. Taken together, these studies provided evidence for a role of GPR35 in mediating IDO1 immune suppression by regulating Hippo-YAP pathway in Treg and CTL cells in the immune system. Thus, GPR35 inhibitors have a potential to be novel immuno-therapeutic agents effective in treating patients with IDO1-positive tumors. Reference1.Munn, DH, and Mellor, AL. (2007) Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J. Clin. Invest. 117:1147-11542.Ni, X. et al. Yap is essential for Treg-mediated suppression of antitumor Immunity. Cancer Discovery 2018; 8:1026-10433.Lebid, A. et al. (2020) YAP attenuates CD8 T cell-mediated anti-tumor response. Frontiers in Immunology 11:1-84.Stampouloglou, E. et al. (2020) YAP suppresses T-cell function and infiltration in the tumor microenvironment. PLoS Biol 18(1): e30005915.Fallarino, F. et al. (2006) The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor æ-chain and induce a regulatory phenotype in naïve T cells. J Immunol 176:6752-6761 Citation Format: Hongmei He, Nam Hee Kim, Sangdon Han, Zhi Liang Chu. Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway [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 LB426.

Intravenous administration of IL-12 encoding self-replicating RNA-lipid nanoparticle complex leads to safe and effective antitumor responses

Interleukin 12 (IL-12) is a potent immunostimulatory cytokine mainly produced by antigen-presenting cells (e.g., dendritic cells, macrophages) and plays an important role in innate and adaptive immunity against cancers. Therapies that can synergistically modulate innate immunity and stimulate adaptive anti-tumor responses are of great interest for cancer immunotherapy. Here we investigated the lipid nanoparticle-encapsulated self-replicating RNA (srRNA) encoding IL-12 (referred to as JCXH-211) for the treatment of cancers. Both local (intratumoral) and systemic (intravenous) administration of JCXH-211 in tumor-bearing mice induced a high-level expression of IL-12 in tumor tissues, leading to modulation of tumor microenvironment and systemic activation of antitumor immunity. Particularly, JCXH-211 can inhibit the tumor-infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). When combined with anti-PD1 antibody, it was able to enhance the recruitment of T cells and NK cells into tumors. In multiple mouse solid tumor models, intravenous injection of JCXH-211 not only eradicated large preestablished tumors, but also induced protective immune memory that prevented the growth of rechallenged tumors. Finally, intravenous injection of JCXH-211 did not cause noticeable systemic toxicity in tumor-bearing mice and non-human primates. Thus, our study demonstrated the feasibility of intravenous administration of JCXH-211 for the treatment of advanced cancers.

Abstract 1087: Synergistic mechanisms against pediatric bone sarcoma models: Trabectedin enhances oncolytic virotherapy intratumoral spread and antitumor immune activation

Abstract Osteosarcoma and Ewing sarcoma are the most common pediatric bone malignancies and, despite decades of research, efforts to treat patients with advanced disease remain dismal. To address this inadequacy, we leverage oncolytic herpes simplex virotherapy as a double-edged attack against tumors through 1) tumor cell-specific lysis and 2) antitumor immune stimulation. However, previous trials of oncolytic herpes simplex virotherapy in pediatric sarcomas displayed limited responses, likely due to the observed presence of immunosuppressive monocytes and macrophages. To improve efficacy, we combined oncolytic herpes simplex virotherapy with trabectedin, an FDA-approved DNA-binding agent known to disrupt gene expression and deplete monocytes and macrophages. Surprisingly, we found that the efficacy of this combination far surpassed our predictions, inducing complete and partial regressions in multiple models of Ewing sarcoma and osteosarcoma. To explore potential mechanisms driving this high degree of synergy, we performed single cell RNA sequencing on tumors of mice treated with monotherapy or combination therapy. Interestingly, trabectedin synergized with the oncolytic virotherapy through two unexpected mechanisms. Our analysis revealed that trabectedin increased intratumoral oncolytic virus spread by disrupting expression of the intrinsic antiviral response in human Ewing sarcoma cells. Additionally, trabectedin not only reduced immunosuppressive macrophages but also stimulated cytotoxic granzyme expression in infiltrating T and NK cells in immunocompetent osteosarcoma models. These effector NK and T cells were necessary for the regressions of osteosarcoma allografts treated with trabectedin and oncolytic virotherapy. Thus, trabectedin enhanced both the oncolytic effect of herpes simplex virotherapy and the effector function of cytotoxic lymphocytes that become activated in response to the virus. The combination of trabectedin and oncolytic herpes simplex virotherapy offers the potential to unite these mechanisms against bone tumors to achieve better outcomes for children. Citation Format: Emily Ringwalt, Mark Currier, Andrea Glaspell, Matthew V. Cannon, Chun-Yu Chen, Amy C. Gross, Maren Cam, Pin-Yi Wang, Ryan D. Roberts, Timothy P. Cripe. Synergistic mechanisms against pediatric bone sarcoma models: Trabectedin enhances oncolytic virotherapy intratumoral spread and antitumor immune activation [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 1087.

Abstract 6685: Control of melanoma development by B. rodentium in germ free mice

Abstract Growing evidence supports the importance of the gut microbiota in controlling tumor development, often via activation of anti-tumor immunity. Earlier studies from our laboratory identified 11 bacterial strains that were sufficient to inhibit melanoma development by inducing anti-tumor immunity in germ-free mice. Of these, three were predominant at the time of tumor collection. We thus set out to address whether fewer bacterial strains may be sufficient for melanoma growth inhibition. Here, we demonstrate that the administration of one bacterial strain, B. rodentium, was sufficient to attenuate melanoma growth in gnotobiotic mice. In all cases, gnotobiotic mice were also administered the altered Schadler flora (ASF), a mixture of 8 bacterial strains that are provided to establish minimal flora in these mice. RNAseq-based gene expression studies identified enrichment of immune-related genes, suggesting enhanced anti-tumor immunity. Metabolomic analysis identified reduced levels of tryptophane and isoleucine, which were implicated in control of immune system function. These findings point to a mechanism which may provide novel means to alter anti-tumor immunity and limit melanoma growth. Citation Format: Ximena Diaz Olea, Kristin Beede, Andrei Osterman, David Scott, Christopher Petucci, Daniel Kelly, Amanda Ramer-Tait, Ze’ev A. Ronai. Control of melanoma development by B. rodentium in germ free mice [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 6685.

Abstract 6690: Gut microbial metabolite butyrate facilitates antitumor efficacy of telomerase-specific oncolytic adenovirus via MHC-I and cGAS-STING pathway activation

Abstract Introduction: Gut microbiota has been reported to have a potential to modulate tumor sensitivity to chemo- or immuno-therapies although the underlying mechanism is still unclear. Oncolytic virotherapy is a promising antitumor treatment that selectively induces oncolytic cell death in tumor cells. We have previously developed OBP-702, a telomerase-specific oncolytic adenovirus armed with the p53 tumor suppressor gene, highlighting potent antitumor effects across different cancer types. To explore the potential of gut microbial metabolites, particularly butyrate, in augmenting the antitumor efficacy of oncolytic virotherapy. Materials and Methods: We assessed the synergistic antitumor effects of butyrate and OBP-702 in cytotoxic assay using two human (HCT116, SW48) and two murine (CT26, MC38) colorectal cancer cell lines in vitro, and the in vivo antitumor effects in subcutaneous tumor models using HCT116 and CT26. We explored underlying mechanisms of the synergistic effects, focusing on the influences of butylate on viral infectivity via Coxsackie-Adenovirus Receptor (CAR) and integrins, and antitumor immunity via tumor MHC-I expression and CD8-positive T cells. Results: Butyrate and OBP-702 showed potent synergistic effects in all four cell lines in vitro, and the combination suppressed tumor growth significantly in HCT116 and CT26 subcutaneous tumors compared to each monotherapy. These synergistic effects were produced by butyrate increasing the potential of OBP-702 by enhancing viral infection efficiency via upregulating expressions of CAR and integrins on tumor cells. Butyrate also increased MHC-I expression on tumor cells by activating the cGAS-STING pathway, leading to increased CXCL10 expression, which led to activation of antitumor immunity through recruitment of CD8-positive T cells in tumor tissues. The synergistic effects of butyrate and OBP-702 were proved in an orthotopic colorectal tumor model with liver metastases using CT26 cells expressing luciferase, in which this combination not only reduced tumor growth, but also improved survival by activating antitumor immunity via CD8-positive T cells. Conclusion: Butyrate and OBP-702 showed synergistic antitumor effects via activation of systemic antitumor immunity in addition to direct effects of butyrate on viral cytotoxic potential, which provides valuable insights into the development of innovative cancer treatment strategies. Citation Format: Masaki Sakamoto. Gut microbial metabolite butyrate facilitates antitumor efficacy of telomerase-specific oncolytic adenovirus via MHC-I and cGAS-STING pathway activation [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 6690.

Abstract 6652: Advanced glioblastoma immunotherapy: Attenuated herpes oncolytic virus armed with anti-PD-1 antibody and IL-12

Abstract Background: Glioblastoma (GBM), the most common and deadly primary brain tumor in adults, has limited treatment options with poor outcomes. The urgent need for innovative treatments has spurred research into immunotherapy and oncolytic virus therapy as promising alternatives. Herein, we have developed a new generation oncolytic herpes simplex virus (oHSV) armed with IL-12 and anti-PD-1 antibody to provide a synergistic anti-GBM efficacy for immune-oncolytic therapy. Methods: MVR-C5252, a novel oHSV, was created by genetic engineering, which included the removal of a 15-kb internal repeat (IR) region and both copies of the γ34.5 genes, along with the insertion of two exogenous genes encoding the anti-PD-1 antibody and IL-12. MVR-C5252 was characterized and assessed for its anti-GBM activities through a series of experiments as follows: 1) Safety assessments, including evaluations of neurotoxicity and latency-reactivation characteristics, were conducted in Balb/C mice. 2) Anti-tumor activities were evaluated in cell cultures, subcutaneous and orthotopic xenograft mouse models, and immunocompetent mouse models. 3) Mechanistic studies were performed to uncover the mode of action of MVR-C5252, with a specific focus on its ability to induce cell death in GBM cells and activate the immune response in vivo. Results: 1) In vitro studies showed that MVR-C5252 is highly replication attenuated but with higher cell-killing activity in GBM cells compared to the first generation oHSV, R3616, which has γ34.5 deletions only. 2) Mechanistic studies showed that MVR-C5252 infection increased cell death by reducing CNTNRα expression, which typically prevents caspase 3 activation and has an anti-apoptotic effect in glioma cells. 3) Intracranial injection of MVR-C5252 revealed a remarkable safety profile, with over an 800-fold reduction in virulence compared to wild-type HSV-1. Furthermore, the mouse trigeminal ganglion (TG) infection model suggested that MVR-C5252 was unable to establish latency or reactivate in the TG. 4) In vivo subcutaneous and orthotopic xenograft mouse models showed that compared to the R3616 group, enhanced anti-tumor activity and extended survival were observed in the MVR-C5252 group. Additionally, in the syngeneic mice model, MVR-C5252 exhibited superior anti-tumor activity and immune activation over the backbone virus. Conclusions: With the unique combination of safety, potent anti-tumor activity, and immune-stimulatory properties, MVR-C5252 makes it a big potential candidate to address the pressing need for effective treatment strategies of GBM. Citation Format: Lei Wang, Xusha Zhou, Xiaoqing Chen, Yuanyuan Liu, Yanxin Zheng, Runbin Yan, Yonghong Liu, Jing Zhao, Grace Guoying Zhou. Advanced glioblastoma immunotherapy: Attenuated herpes oncolytic virus armed with anti-PD-1 antibody and IL-12 [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 6652.

Abstract 2700: Therapeutic IL-33 enhances the antigen presentation function of ILC2s to exert anti-tumor immune activity

Abstract IL-33 regulates the function of ILC2s through ST2. However, the role of IL-33, especially exogenous IL-33, on ILC2s in tumors is controversial depending on the cytokines they secrete. Here we report that exogenous, but not endogenous, IL-33 significantly inhibits the progression of a variety of mouse solid tumors, including melanoma, colorectal cancer, lung adenoma, and breast cancer. By inoculating tumor cells in Il1rl1−/- and NSG mice, we found that the tumor-suppressive effect of IL-33 on tumors was dependent on the expression of ST2 receptors and the presence of lymphocytes. Regarding tumor-infiltrating immune cells, exogenous IL-33 significantly increases the proportions of ST2+ Tregs (1.10 ± 0.20% vs. 1.74 ± 0.36%), eosinophils (6.00 ± 1.33% vs. 22.73 ± 6.51%), Granzyme B+ CD8+ T cells (4.32 ± 2.77% vs. 11.53 ± 6.81%), and bulk ILC2s (0.08 ± 0.04% vs. 5.31 ± 1.33%) and IA/IE+ ILC2s (0.02 ± 0.01% vs. 3.67 ± 1.03%). In draining lymph nodes, IL-33 also significantly increases the proportions of ILC2s (0.03 ± 0.02‰ vs. 6.37 ± 1.71‰) and IA/IE+ ILC2s (0.003 ± 0.003‰ vs. 4.38 ± 1.21‰). Importantly, single-cell transcriptomic analysis on tumor-infiltrating ILC2s highlights the expression of genes related with antigen processing and presentation including H2-K1, H2-Q7, B2m, Hspa1a, Hspa1b, Creb1, Hspa4, Tap1 and Tap2. We testified this finding by DQ-OVA experiment and found that IL-33 could promote ILC2s to up-take and process exogenous antigens (17.78 ± 0.75 fold vs. 22.02 ± 0.75 fold). The antigen presentation function of ILC2s was further established by mixed lymphocyte culture where BALB/c ILC2s potently stimulated C57BL/6 CD8+ T cell proliferation, which was further augmented by IL-33 treatment. Taken together, our data demonstrate an anti-tumor function of therapeutic IL-33 that elevates the number and antigen presentation function of ILC2s. Citation Format: Jie Liu, Zhenchu Feng, Hongyan Wei, Chang Li, Xiaoshi Li, Wenlong Chen, Yixi Ke, Minjun Wang, Lantian Liu, Cunni Zheng, Quan Liu. Therapeutic IL-33 enhances the antigen presentation function of ILC2s to exert anti-tumor immune activity [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 2700.

Abstract 2472: Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models

Abstract Background: ENPP1, a nucleotide pyrophosphatase and phosphodiesterase, has emerged as an important modulator of anti-tumor immune activation, as it regulates the concentrations of cGAMP and ATP in the TME. ENPP1 expression in tumors is common and correlates with a cold tumor phenotype. Its loss has been reported to suppress metastasis, restore immune cell infiltration into the tumor and potentiate the response to immune checkpoint blockade, while, conversely, the overexpression of ENPP1 promoted metastasis and rendered tumors resistant to checkpoint blockade. RBS2418 was designed as a potent and selective inhibitor of ENPP1 and is in clinical development. Methods: ENPP1 enzyme inhibition was measured in vitro using cGAMP and ATP substrates. Effects of pH and serum and selectivity against related enzymes were evaluated. In vivo efficacy was assessed in Hepa1-6, and GL261-luc syngeneic liver and glioblastoma mouse models. Compound exposures were determined in tumor and blood samples. Cures were confirmed by re-challenge of cured animals with the same cancer cells. Results: RBS2418 inhibited hydrolysis of cGAMP and ATP in vitro with similar potency (Ki =0.14 and 0.13 nM). Inhibition was also similar in human serum, consistent with low binding affinity to human serum albumin. In humans, oral dosing at doses of 100 mg could inhibit ENPP1 enzyme activity during the full dosing interval. Dosing of RBS2418 was performed in mice to achieve trough concentrations of RBS2418 in tumors and plasma exceeding ENPP1 EC90 levels for the complete dosing interval. In the Hepa1-6 model treatment with RBS2418 for either 2 or 10 days resulted in significant reduction of tumor growth and prolongation of survival as compared to vehicle control. There was no significant difference between the RBS2418 groups. Complete tumor regression was observed in 44% of treated mice across the two treatment groups. These mice were resistant to rechallenge with Hepa1-6 tumor cells. In the GL261-luc model treatment with RBS2418 showed significant reduction of tumor burden and prolongation of survival as compared to vehicle control. The mice that achieved complete tumor regression were resistant to rechallenge with GL261-luc tumor cells. Conclusions: RBS2418 inhibited ATP and cGAMP hydrolysis by ENPP1 with similar high potency. The pharmacology profile was optimized to achieve low serum protein binding and oral bioavailability in humans. RBS2418 treatment was well tolerated in mice under conditions where tumor and plasma levels exceeding ENPP1 serum EC90 levels were achieved. In the syngeneic mouse models Hepa1-6 and GL261-luc significant tumor growth reductions, complete tumor regression and cures were observed. Resistance to cancer cell rechallenge was consistent with treatment-induced anti-tumor immunity. Citation Format: Ningwu Huang, Junjun Cheng, Ron Hawley, Klaus Klumpp. Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models [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 2472.