Increased T-cell Infiltration Research Articles

T cell-Mediated Development of Stromal Fibroblasts with an Immune-Enhancing Chemokine Profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAF) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA sequencing of CAFs from mouse pancreatic adenocarcinomas identified a subpopulation of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune-suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

Supramolecular Filament Hydrogel as a Universal Immunomodulator Carrier for Immunotherapy Combinations.

A major challenge of cancer immunotherapy is to develop delivery strategies that can effectively and safely augment the immune system's antitumor response. Here, we report on the design and synthesis of a peptide-based supramolecular filament (SF) hydrogel as a universal carrier for localized delivery of three immunomodulating agents of distinct action mechanisms and different molecular weights, including an aPD1 antibody, an IL15 cytokine, and a STING agonist (CDA). We show that in situ hydrogelation can be triggered to occur upon intratumoral injection of SF solutions containing each of aPD1, IL15, or CDA. The formed hydrogel serves as a scaffold depot for sustained and MMP-2-responsive release of immunotherapeutic agents, achieving enhanced antitumor activities and reduced side effects. When administered in combination, the aPD1/IL15 or aPD1/CDA hydrogel led to substantially increased T-cell infiltration and prevented the development of adaptive immune resistance induced by IL15 or CDA alone. These immunotherapy combinations resulted in complete regression of established large GL-261 tumors in all mice and elicited a protective long-acting and systemic antitumor immunity to prevent tumor recurrence while eradicating distant tumors. We believe this SF hydrogel offers a simple yet generalizable strategy for local delivery of diverse immunomodulators for enhanced antitumoral response and improved treatment outcomes.

Sequential treatment with a TNFR2 agonist and a TNFR1 antagonist improves outcomes in a humanized mouse model for MS

TNF signaling is an essential regulator of cellular homeostasis. Through its two receptors TNFR1 and TNFR2, soluble versus membrane-bound TNF enable cell death or survival in a variety of cell types. TNF-TNFRs signaling orchestrates important biological functions such as inflammation, neuronal activity as well as tissue de- and regeneration. TNF-TNFRs signaling is a therapeutic target for neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer’s disease (AD), but animal and clinical studies yielded conflicting findings. Here, we ask whether a sequential modulation of TNFR1 and TNFR2 signaling is beneficial in experimental autoimmune encephalomyelitis (EAE), an experimental mouse model that recapitulates inflammatory and demyelinating aspects of MS. To this end, human TNFR1 antagonist and TNFR2 agonist were administered peripherally at different stages of disease development in TNFR-humanized mice. We found that stimulating TNFR2 before onset of symptoms leads to improved response to anti-TNFR1 therapeutic treatment. This sequential treatment was more effective in decreasing paralysis symptoms and demyelination, when compared to single treatments. Interestingly, the frequency of the different immune cell subsets is unaffected by TNFR modulation. Nevertheless, treatment with only a TNFR1 antagonist increases T-cell infiltration in the central nervous system (CNS) and B-cell cuffing at the perivascular sites, whereas a TNFR2 agonist promotes Treg CNS accumulation. Our findings highlight the complicated nature of TNF signaling which requires a timely balance of selective activation and inhibition of TNFRs in order to exert therapeutic effects in the context of CNS autoimmunity.

Abstract CT206: A phase I study of binimetinib, a MEK inhibitor, in combination with pembrolizumab in patients with advanced non-small cell lung cancer (NSCLC)

Abstract Introduction: MEK inhibition combined with PD-1 axis inhibition may achieve a greater clinical response than either inhibitor alone due to increased T-cell infiltration of tumours. We combined binimetinib with pembrolizumab in patients with stage IV advanced non-small cell lung cancer (NSCLC) and PD-L1 tumor proportion score>=50%. Methods: A 3 + 3 dose escalation design was used. Binimetinib at a dose level 1 (DL1; 45 mg) or dose level -1 (DL-1; 30 mg) twice daily orally continuously was given with pembrolizumab 200 mg IV q 21 days. The primary objective was to define the recommended phase II dose (RP2D) of the combination. Secondary outcomes included safety of the combination and response (RECIST 1.1) with a planned Phase Ib expansion in patients with RAS/RAF/MEK dysregulated tumours via next generation gene sequencing. Genomic markers are being explored in tissue and plasma. Results: Eleven patients (3 DL1, 8 DL-1) were enrolled with the following mutations: 7 with KRAS (3 G12C, 2 G12D, 1 G12V, 1 G12A), 2 BRAF (1 G409A, 1 V600E) and 1 STK11 which acts as a tumor suppressor gene encoding for LKB1. Two of 3 patients at DL1 experienced dose limiting toxicity (DLT) including grade 3 elevated amylase, grade 3 diarrhea, grade 4 elevated lipase and severe grade 2 rash requiring dose reduction. Of 8 patients treated at DL-1, 1 progressed in cycle 1, another was noncompliant with treatment. Of the remaining 6 patients, 1 experienced DLT with grade 3 rash with inability to administer binimetinib for >75% of cycle 1. The most common toxicities across all cycles (n=11 patients) were: rash (82%), diarrhea (36%) and pruritis (36%). Nine patients were evaluated for response with partial response in 3 (33%), stable disease in 4 (44%) and progressive disease in 2 (22%). All 3 responding patients had RAS or RAF alterations (KRASG12C, KRASG12V, BRAFV600E) while the patient with STK11 mutant disease had early disease progression. Conclusion: The RP2D of the combination in patients with advanced NSCLC is binimetinib 30 mg BID plus pembrolizumab 200 mg IV q21 days. Updated data from cohort expansion including response and molecular correlates of treatment response and resistance will be presented. Citation Format: Christopher F. Theriau, Jamie Feng, Lawson Eng, Frances A. Shepherd, Mary DeCarolis, Vivian Glenns, Swati Kulkarni, Rachel VanderMeer, Urszula Zurawska-Fortin, Desiree Hao, Lisa Le, Isabel Wozniczka, Aida Al-Kindy, Danny Xie, Tong Zhang, Vasanth Subramanian, Tracy Stockley, Penelope A. Bradbury, Geoffrey Liu, Natasha B. Leighl. A phase I study of binimetinib, a MEK inhibitor, in combination with pembrolizumab in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT206.

Engineering tumor-specific gene nanomedicine to recruit and activate T cells for enhanced immunotherapy

PD-1/PD-L1 blockade therapy that eliminates T-cell inhibition signals is successful, but poor benefits are often observed. Increasing T-cell infiltration and quantity of PD-1/PD-L1 inhibitors in tumor can improve efficacy but remains challenging. Here, we devise tumor-specific gene nanomedicines to mobilize tumor cells to secrete CXCL9 (T-cell chemokine) and anti-PD-L1 scFv (αPD-L1, PD-L1 blocking agent) for enhanced immunotherapy. The tyrosinase promoter-driven NPTyr-C9AP can specifically co-express CXCL9 and αPD-L1 in melanoma cells, thereby forming a CXCL9 gradient for T-cell recruitment and high intratumoral αPD-L1 concentration for enhancing T-cell activation. As a result, NPTyr-C9AP shows strong antimelanoma effects. Moreover, specific co-expression of CXCL9 and αPD-L1 in various tumor cells is achieved by replacing the tyrosinase promoter of NPTyr-C9AP with a survivin promoter, which increases T-cell infiltration and activation and therapeutic efficacy in multiple tumors in female mice. This study provides a strategy to maximize the immunotherapeutic outcome regardless of the heterogeneous tumor microenvironment.

Abstract 3643: PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer

Abstract This study aims to test the hypothesis that direct interactions and auto-regulatory loop between PTEN and STAT3 in cancer-associated fibroblasts (CAFs) contribute to an immunosuppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). Pancreatic cancer accounts for almost as many deaths as cases because of its poor prognosis. Many studies have led to the paradigm that stromal elements contribute to pancreatic tumor growth. Cancer-associated fibroblasts (CAFs) are a primary cell type in the desmoplastic stroma in PDAC, and targeting them to improve PDAC treatment is thought to be a promising strategy, however; studies have shown both tumor-promoting and tumor-limiting activities associated with PDAC CAFs. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most well-described tumor suppressor genes and can regulate essential transcription factors in stromal fibroblasts. On the other hand, STAT3 signaling in pancreatic CAFs contributes to the immunosuppressive and fibrotic stroma observed with disease progression. In this study, we used novel, dual recombinase mouse models of PDAC, with Kras and p53 mutations engineered in the pancreatic epithelium (Flp-based) and Stat3 deleted in a subpopulation of CAFS (Cre). CAFs isolated from these mice were used to probe PTEN-STAT3 interaction in vitro using the Proximity Ligation Assay (PLA) and by ChIP-seq using an RNA seq. Mouse and Human pancreatic tumor samples were analyzed and quantified using multiplex-immunofluorescence and multispectral imaging. In the mouse genetic model, we found PTEN downregulated in ~80% of CAFs, consistent with previous work from our group that demonstrated PTEN loss in 25% of PDAC patient samples. At the same time, pY705-STAT3 was activated in CAFs, and deletion of STAT3 in CAFs correlated with increased survival, decreased M2-like macrophages, and increased T-cell infiltration. STAT3 ablation in CAFS resulted in the re-expression of PTEN in CAFS, implying an autoregulatory loop. PLA on CAFs isolated revealed that IL6 treatment resulted in the transfer of a STAT3-PTEN protein complex into the nucleus ChIP-seq on CAFs with PTEN STAT3 antibodies revealed a significant overlap in PTEN and STAT3 occupation of gene promoters. This result is consistent with in silico analysis on the PTEN and STAT3 ChIP-Seq data in human cell lines. ChIP-Seq data is being interacted with RNA data to identify essential target genes. Human PDAC tissue microarrays are stained with pSTAT3, PTEN, CD3, and CD8.Our study suggests an autoregulatory look between PTEN and STAT3 in PDAC CAFs. PTEN may suppress specific STAT3 target genes through direct interactions with chromatin. Alternatively, PTEN/STAT3 complexes may target CAF genes that suppress tumor growth or activate anti-tumor immune responses. These investigations could identify potential targets in CAFs for restoring the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Sudarshana Sharma, Cameron Bumbleburg, Julia E. Lefler, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3643.

Abstract 6425: Functional roles of enhancers in immune microenvironment & immunotherapy response

Abstract Immune checkpoint blockade (ICB) therapy has improved long-term survival for patients with advanced melanoma. However, there is critical need to identify potential biomarkers of response and actionable strategies to improve response rates. Through generation and analysis of over 200 chromatin modification maps for ICB-treated melanoma patient samples, melanoma cells and T cells, we identified significant enrichment of active enhancer states in non-responders at baseline. Enhancer mapping by bulk ChIP-Seq or single cell ATAC-Seq methods in two independent cohorts of ICB-treated melanoma samples identified an enhancer signature that predicted response to anti-PD-1 therapy. The activated non-responder enhancers marked a group of key regulators of several pathways in melanoma cells (including c-MET, TGFβ, EMT and AKT) that are known to mediate resistance to ICB therapy. In addition, several checkpoint receptors were alternatively activated by aberrant enhancers in T cells. Unbiased CRISPRi screening in melanoma cells and T cells identified novel functional enhancers, such as one upstream of c-MET, that mediate ICB response or T cell mediated killing. Finally, inhibition of enhancers and repression of these pathways using bromodomain inhibitors along with anti-PD-1 therapy significantly decreased melanoma tumor burden and increased T-cell infiltration. Epigenomic experiments identified a signature of 107 genes that could be used as pharmacodynamic marker for BET inhibitor response. Together, these findings identify enhancer upregulation as a key mechanism of adaptive resistance to ICB response, a potential enhancer-based biomarker of resistance to anti-PD-1 and enhancer blockade in combination with ICB as a potential strategy to improve responses. Citation Format: Kunal Rai, Mayinuer Maitituoheti, Alvin Shi, Ming Tang, Li-Lun Ho, Firas Youssef Kreidieh, Christopher Terranova, Kyriakitsa Galani, Emily Z. Keung, Caitlin A. Creasy, Manrong Wu, Jiajia Chen, Nana Chen, Anand K. Singh, Apoorvi Chaudhri, Nazanin E. Anvar, Giuseppe Tarrantino, Jiekun Yang, Sharmistha Sarkar, Shan Jiang, Jared Malke, Lauren Haydu, Elizabeth Burton, Michael A. Davies, Jeffrey E. Gershenwald, Patrick Hwu, Alexander Lazar, Jaime H. Cheah, Christian K. Soule, Stuart S. Levine, Chantale Bernatchez, Srinivas V. Saladi, David Liu, Hussein Tawbi, Jennifer Wargo, Genevieve M. Boland, Manolis Kellis. Functional roles of enhancers in immune microenvironment & immunotherapy response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6425.

Abstract 6800: Unique autologous cancer vaccine comprised of irradiated whole tumor cells and MBTA (rWTC-MBTA) triggers antitumor immune response to prevent metastasis

Abstract Despite the advances made in cancer treatment over the past decades, one statistic has remained unchanged: metastatic cancer accounts for 90 percent of annual cancer deaths in the United States. Our group previously demonstrated that an autologous whole tumor cell vaccine (rWTC-MBTA: irradiated autologous whole tumor cells pulsed with Mannan-BAM, TLR ligands, and anti-CD40 antibody) had a potent anti-tumor immune response and prolonged survival in a mouse colon carcinoma model. To investigate whether rWTC-MBTA would work in “cold” tumor models, we evaluated the vaccine’s effect on preventing and treating tumor metastasis in 4T1 breast tumors. Our data showed that vaccinated mice could significantly prevent lung metastasis in both intravenous injection and mammary pad subcutaneous implantation animal models. Further, we used another metastasis model that more closely mimics clinical practice by resecting the primary tumor after metastasis development. The data showed that vaccinated mice could prevent tumor recurrence, increase T-cell infiltration in the metastatic tumor, and prolong the survival curve. The mechanistic investigation by immunophenotyping revealed that rWTC-MBTA vaccination induced both effector memory (CD44+CD62L−) and center memory (CD44+CD62L+) T cells as well as increased overall CD4+ and CD8+ T-cell count while depletion experiments demonstrated that CD8+ T cells were required for vaccine efficacy. Isolated splenocytes co-cultured with 4T1 cells showed rWTC-MBTA significantly increased T-cell mediated cytotoxicity through TNF-a and IFN-γ in CD107a+CD4+ T cells and Granzyme B and IFN-γ in CD107a+CD8+ T cells. In all experiments, vaccination exerted negligible systemic toxicity. Collectively, our study demonstrates that the rWTC-MBTA vaccine is a safe and promising therapeutic option to prevent and treat tumor metastasis by triggering an antitumor immune response. Citation Format: Juan Ye, Herui Wang, Mitchell Sun, Ondrej Uher, Mark R. Gilbert, Karel Pacak, Zhengping Zhuang, Rogelio Medina, Samik Chakraborty, Jan Zenka. Unique autologous cancer vaccine comprised of irradiated whole tumor cells and MBTA (rWTC-MBTA) triggers antitumor immune response to prevent metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6800.

Tumor-targeted superantigens produce curative tumor immunity with induction of memory and demonstrated antigen spreading

BackgroundDespite remarkable progress, the immunotherapies currently used in the clinic, such as immune checkpoint blockade (ICB) therapy, still have limited efficacy against many types of solid tumors. One major barrier to effective treatment is the lack of a durable long-term response. Tumor-targeted superantigen (TTS) therapy may overcome this barrier to enhance therapeutic efficacy. TTS proteins, such as the clinical-stage molecule naptumomab estafenatox (NAP), increase tumor recognition and killing by both coating tumor cells with bacterial-derived superantigens (SAgs) and selectively expanding T-cell lineages that can recognize them. The present study investigated the efficacy and mechanism of action of repeated TTS (C215Fab-SEA) treatments leading to a long-term antitumor immune response as monotherapy or in combination with PD-1/PD-L1 inhibitors in murine tumor models.MethodsWe used syngeneic murine tumor models expressing the human EpCAM target (C215 antigen) to assess the efficacy and mechanism of action of repeated treatment with TTS C215Fab-SEA alone or with anti-PD-1/PD-L1 monoclonal antibodies. Tumor draining lymph nodes (TDLNs) and tumor tissues were processed and analyzed by immunophenotyping and immunohistochemistry. Isolated RNA from tumors was used to analyze gene expression and the TCR repertoire. Tumor rechallenge and T-cell transfer studies were conducted to test the long-term antitumor memory response.ResultsTTS therapy inhibited tumor growth and achieved complete tumor rejection, leading to a T-cell-dependent long-term memory response against the tumor. The antitumor effect was derived from inflammatory responses converting the immunosuppressive TME into a proinflammatory state with an increase in T-cell infiltration, activation and high T-cell diversity. The combination of TTS with ICB therapy was significantly more effective than the monotherapies and resulted in higher tumor-free rates.ConclusionsThese new results indicate that TTSs not only can turn a “cold” tumor into a “hot” tumor but also can enable epitope spreading and memory response, which makes TTSs ideal candidates for combination with ICB agents and other anticancer agents.

Tumor Cell-Derived Microparticles Induced by Methotrexate Augment T-cell Antitumor Responses by Downregulating Expression of PD-1 in Neutrophils.

Neutrophils act as a "double-edged sword" in the tumor microenvironment by either supporting or suppressing tumor progression. Thus, eliciting a neutrophil antitumor response remains challenging. Here, we showed that tumor cell-derived microparticles induced by methotrexate (MTX-MP) acts as an immunotherapeutic agent to activate neutrophils, increasing the tumor-killing effect of the cells and augmenting T-cell antitumor responses. We found that lactate induced tumor-associated neutrophils to elevate expression of programmed cell death protein 1 (PD-1) and that PD-1+ neutrophils had the properties of N2 neutrophils and suppressed T-cell activation through PD-1/programmed death-ligand 1 (PD-L1) signaling. By performing ex vivo experiments, we found that MTX-MPs-activated neutrophils had reduced surface expression of PD-1 as a result of PD-1 internalization and degradation in the lysosomes, leading to the cells showing a decreased capacity to suppress T-cell responses. In addition, we also found that MTX-MP-activated neutrophils released neutrophil elastase which could kill tumor cells and disrupt tumor stroma, leading to increased T-cell infiltration. Furthermore, using a combination of anti-PD-L1 and MTX-MPs, we observed that long-term survival increased in a mouse model of lung cancer. Collectively, these findings highlight the potential use of a combination of anti-PD-L1 and MTX-MPs to enhance the therapeutic effect of anti-PD-L1 alone.

Reprogramming systemic and local immune function to empower immunotherapy against glioblastoma

The limited benefits of immunotherapy against glioblastoma (GBM) is closely related to the paucity of T cells in brain tumor bed. Both systemic and local immunosuppression contribute to the deficiency of tumor-infiltrating T cells. However, the current studies focus heavily on the local immunosuppressive tumor microenvironment but not on the co-existence of systemic immunosuppression. Here, we develop a nanostructure named Nano-reshaper to co-encapsulate lymphopenia alleviating agent cannabidiol and lymphocyte recruiting cytokine LIGHT. The results show that Nano-reshaper increases the number of systemic T cells and improves local T-cell recruitment condition, thus greatly increasing T-cell infiltration. When combined with immune checkpoint inhibitor, this therapeutic modality achieves 83.3% long-term survivors without recurrence in GBM models in male mice. Collectively, this work unveils that simultaneous reprogramming of systemic and local immune function is critical for T-cell based immunotherapy and provides a clinically translatable option for combating brain tumors.

Lung tumor microphysiological system with 3D endothelium to evaluate modulators of T-cell migration.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation, and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor-on-chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a HUVEC-derived vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells migrate; and a chemoattractant/tumor (HCC0827 or NCI-H520) compartment. Here, activated T-cells extravasate and migrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose translational strategies by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

Moringa oleifera leaf polysaccharides exert anti-lung cancer effects upon targeting TLR4 to reverse the tumor-associated macrophage phenotype and promote T-cell infiltration.

Tumor-associated macrophages (TAMs) participate in tumorigenesis, growth, invasion as well as metastasis by facilitating an immunosuppressive tumor microenvironment. Reversing the pro-tumoral M2 phenotype of TAMs has become a hot spot in advancing cancer immunotherapy. In the current study, the content of Moringa oleifera leaf polysaccharides (MOLP) was determined and characterized, along with the anti-cancer mechanism of MOLP studied in a Lewis lung cancer (LLC) tumor-bearing mouse model and bone marrow-derived macrophages. The monosaccharide composition and gel permeation chromatography analyses show that MOLP are mainly composed of galactose, glucose, and arabinose, with approximately 17.35 kDa average molecular weight (Mw). In vivo studies demonstrate that MOLP convert TAMs from the immunosuppressive M2 phenotype to the antitumor M1 phenotype, thus inducing CXCL9 and CXCL10 expression and increasing T-cell infiltration in the tumor. Furthermore, macrophage depletion and T cell suppression demonstrated that the tumor suppressive effect of MOLP was reliant on reprogramming macrophage polarization and T cell infiltration. In vitro studies revealed that MOLP could induce the phenotypic switch from M2 macrophages to M1 by targeting TLR4. The current study highlights that MOLP are promising anticancer plant-derived polysaccharides with potential in modulating the immune microenvironment and have a bright application prospect in the immunotherapy of lung cancer.

169P Immunomodulatory effects of RBS2418, an oral ENPP1 inhibitor in combination with pembrolizumab in checkpoint-refractory metastatic adrenal cancer

ENPP1 is a type II transmembrane protein with nucleotide pyrophosphatase, and phosphodiesterase enzymatic activities and its expression is associated with poor prognosis in cancer. ENPP1 inhibition protects cGAMP and ATP from hydrolysis and reduces adenosine levels in the TME, activates APCs and increases T-cell infiltration promoting anticancer immunity. RBS2418 is a potent oral ENPP1 inhibitor. Here, we report pharmacokinetic (PK) and immunomodulatory properties of RBS2418 in combination with pembrolizumab (pembro) in a checkpoint-refractory high grade adrenal cancer with an immune-desert tumor phenotype at baseline.

195TiP A first-in-human, phase I a/b dose escalation and expansion study to evaluate RBS2418 as monotherapy and in combination with pembrolizumab in subjects with advanced unresectable, recurrent or metastatic tumors

ENPP1 is a type II transmembrane protein with nucleotide pyrophosphatase, and phosphodiesterase enzymatic activities and its expression is associated with poor prognosis in cancer. ENPP1 inhibition protects cGAMP and ATP from hydrolysis and reduces adenosine levels in the TME, activates APCs and increases T-cell infiltration promoting anticancer immunity. RBS2418 is a potent oral ENPP1 inhibitor. The current study evaluates RBS2418 as monotherapy and combination with pembrolizumab (pembro), supporting preclinical data and complementary mechanism of action with the aim of generating improved anti-tumor immunity and overcoming checkpoint inhibitor resistance in the combination setting.

Polymeric micelles effectively reprogram the tumor microenvironment to potentiate nano-immunotherapy in mouse breast cancer models

Nano-immunotherapy improves breast cancer outcomes but not all patients respond and none are cured. To improve efficacy, research focuses on drugs that reprogram cancer-associated fibroblasts (CAFs) to improve therapeutic delivery and immunostimulation. These drugs, however, have a narrow therapeutic window and cause adverse effects. Developing strategies that increase CAF-reprogramming while limiting adverse effects is urgent. Here, taking advantage of the CAF-reprogramming capabilities of tranilast, we developed tranilast-loaded micelles. Strikingly, a 100-fold reduced dose of tranilast-micelles induces superior reprogramming compared to free drug owing to enhanced intratumoral accumulation and cancer-associated fibroblast uptake. Combination of tranilast-micelles and epirubicin-micelles or Doxil with immunotherapy increases T-cell infiltration, resulting in cures and immunological memory in mice bearing immunotherapy-resistant breast cancer. Furthermore, shear wave elastography (SWE) is able to monitor reduced tumor stiffness caused by tranilast-micelles and predict response to nano-immunotherapy. Micellar encapsulation is a promising strategy for TME-reprogramming and SWE is a potential biomarker of response.

RTID-02. A PHASE 1, SAFETY LEAD-IN AND RANDOMIZED, OPEN-LABEL, PERIOPERATIVE STUDY OF VORASIDENIB COMBINED WITH PEMBROLIZUMAB IN RECURRENT OR PROGRESSIVE ENHANCING IDH-1 MUTANT ASTROCYTOMAS: TRIAL IN PROGRESS

Abstract BACKGROUND Astrocytomas are a subset of diffuse gliomas defined by intact 1p and 19q arms. Isocitrate dehydrogenase 1 mutations (mIDH-1) occur in ~70% of grade 2/3 gliomas, leading to accumulation of 2-hydroxyglutarate (2-HG). Vorasidenib (VOR) is an oral, brain-penetrant dual inhibitor of mIDH1/2 enzymes being investigated in a phase 3 study in non-enhancing gliomas. In an ongoing perioperative study in grade 2/3 gliomas, treatment with VOR was associated with interferon (IFN) signaling activation and increased T-cell infiltration, suggesting adequate 2-HG suppression renders the tumor immune microenvironment for immune checkpoint blockade, and supporting investigation of VOR in combination with an anti-programmed cell death (PD-1) antibody such as pembrolizumab in recurrent IDH-mutant gliomas treatment. This study will evaluate the safety and tolerability of VOR plus pembrolizumab to determine the recommended combination dose (RCD) of VOR, and evaluate CD3+ T-cell infiltration in tumors following preoperative treatment with the combination or VOR. METHODS This study will enroll ~70 patients with recurrent or progressive Grade 2/3 mIDH-1 astrocytoma. Key eligibility: enhancing disease, mIDH1-R132H, Karnofsky Performance Status ≥70, eligible for resection (perioperative phase only). Safety lead-in: Cohort 1 (Nf~6): VOR 40 mg QD plus pembrolizumab 200 mg Q3W in 21-day cycles. Based on DLT evaluation, cohort 2 may enroll an additional ~6 patients at VOR 20 mg QD plus pembrolizumab 200 mg Q3W. Randomized, perioperative phase (Nf~60): randomized 1:1:1 to the combination, VOR 40 mg QD, or untreated for 4 weeks preoperatively; all patients can opt to receive the combination postoperatively. Primary objectives: safety and tolerability, and VOR RCD with pembrolizumab; CD3+ T-cell infiltration in resected tumors following presurgical treatment with the combination compared to untreated tumors. Secondary objectives include clinical activity, VOR PK in tumor and blood, 2-HG concentration in tumors, and overall survival. This study will be active in the United States.

Sex Differences in Genomic Features of Hepatitis B–Associated Hepatocellular Carcinoma With Distinct Antitumor Immunity

Aflatoxin exposure increases the risk for hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected individuals, particularly males. We investigated sex-based differences in the HCC genome and antitumor immunity. Whole-genome, whole-exome, and RNA sequencing were performed on 101 HCC patient samples (47 males, 54females) that resulted from HBV infection and aflatoxin exposure from Qidong. Androgen on the expression of aflatoxin metabolism-related genes and nonhomologous DNA end joining (NHEJ) factors were examined in HBV-positive HCC cell lines, and further tested in tumor-bearing syngeneic mice. Qidong HCC differed between males and females in genomic landscape and transcriptional dysfunction pathways. Compared with females, males expressed higher levels of aflatoxin metabolism-related genes, such as AHR and CYP1A1, and lower levels of NHEJ factors, such as XRCC4, LIG4, and MRE11, showed a signature of up-regulated type I interferon signaling/response and repressed antitumor immunity. Treatment with AFB1 in HBV-positive cells, the addition of 2 nmol/L testosterone to cultures significantly increased the expression of aflatoxin metabolism-related genes, but reduced NHEJ factors, resulting in more nuclear DNA leakage into cytosol to activate cGAS-STING. In syngeneic tumor-bearing mice that were administrated tamoxifen daily via oral gavage, favorable androgen signaling repressed NHEJ factor expression and activated cGAS-STING in tumors, increasing T-cell infiltration and improving anti-programmed cell death protein 1 treatment effect. Androgen signaling in the context of genotoxic stress repressed DNA damage repair. The alteration caused more nuclear DNA leakage into cytosol to activate the cGAS-STING pathway, which increased T-cell infiltration into tumor mass and improved anti-programmed cell death protein 1 immunotherapy in HCCs.

JS08.7.A Integrative analysis of longitudinal genomic and non-genetic mechanisms of radiotherapy-resistance in glioma

Abstract Background Radiotherapy (RT) is a mainstay in the treatment of glioma patients, but RT-resistance is common. We have recently identified RT-associated genomic small deletion signatures, marking tumors that have developed resistance. Here, we used longitudinal molecular profiles to explore non-genetic mechanisms of RT-resistance. Material and Methods We have analyzed the multi-omic Glioma Longitudinal Analysis (GLASS) dataset, integrating DNA and RNA sequencing datasets from primary and post-treatment gliomas. Results We classified tumors as harboring temozolomide-associated hypermutation based on mutational burden increase (HM), and RT-associated signature based on small deletion burden increase (RTscars). By deconvoluting RNA profiles into cell state fractions, we observed an increase of a proliferating stem-like (PSL) cell state in 50% of patients with a RTscars+ signature which was significantly higher than RTscars-/HM- patients (P=8e-04, Fisher’s exact). PSL cell state increase associated with worse overall survival outcomes (P=1.5e-03, log-rank). We observed a significant correlation between the expression change of E2F cell cycle regulator genes (R=0.91, P=6.7e-04, Pearson) and EZH2 (R=0.81, P=8.3e-03) with the increase in the PSL cell state, nominating the E2F/EZH2-pathway as regulator of the PSL cell state. Furthermore, the RTscars signature was associated with an increase in frameshift neoantigens and significantly higher neoantigen burden at recurrence (P=2.4e-02, Kruskal-Wallis). This was accompanied by a significantly higher post-treatment T-cell fraction in RTscars+ tumors (P=8.2e-04, Wilcox), suggesting an increased T-cell infiltration in these patients. Conclusion Our analyses revealed a longitudinal increase in the proliferating stem-like cell state associated with RT-resistance and nominates the cell cycle pathway as an actionable target. The RT-associated deletion signature (RTscars) correlated with increased frameshift-neoantigens and T-cell fractions at recurrence, suggesting a potential benefit of a combinatorial immune-targeted therapy in this specific patient population.

SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice.

The COVID-19 pandemic has been fueled by SARS-CoV-2 novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strain’s pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta-challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing challenged to no challenge mice revealed that Alpha-challenged mice have more total genes differentially activated. Conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-γ production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines.