Vile, Richard G.

Abstract Background: Despite the success of chimeric antigen receptor (CAR) T cell therapy in hematological malignancies, its efficacy in solid tumors remains limited. Macrophages, as key players in the innate immune response, offer unique advantages in the solid tumor context due to their ability to infiltrate tissues and phagocytose tumor cells. Preclinical studies have demonstrated that CAR-engineered macrophages (CAR-Ms) can penetrate solid tumors, initiate phagocytosis, and promote adaptive immunity. Early-phase clinical trials infusing CAR-Ms into patients have shown a favorable safety profile. However, within the immunosuppressive tumor microenvironment (TME), macrophages are often reprogrammed into tumor-promoting, M2-like tumor-associated macrophages (TAMs), which limit their therapeutic efficacy. Metabolic reprogramming is a critical driver of this polarization and TCA derived succinate has been shown to promote M1 phenotype in macrophages. In this study, we investigate whether succinate-loaded nanoparticles (PES NPs) can metabolically rewire CAR-Ms to maintain a pro-inflammatory (M1-like) phenotype and enhance antitumor activity in ovarian cancer (OC). Results: We synthesized polyethylene succinate (PES) nanoparticles (NPs) and analyzed their size, polydispersity, and zeta (ζ) potential using dynamic light scattering (DLS). The results showed a negative potential, indicative of stability, and suggested that succinate could be effectively encapsulated in nanoparticles. OC cells abundantly express mesothelin protein; therefore, mesothelin-targeting CARs were chosen to transfect mouse bone marrow-derived macrophages (BMDMs) or human monocyte-derived macrophages (HMDMs) to generate CAR-Ms. Our results showed that mouse and human CAR-Ms treated with PES NPs exhibited increased mitochondrial respiration and glycolysis, seen as elevated ECAR and OCR relative to vehicle control or cell-permeable dimethyl succinate ester (DMSA). Flow cytometry analysis revealed that PES treatment resulted in increased expression of markers of the classical M1 phenotype in both mouse and human CAR-Ms. The CAR-Ms also had elevated mRNA levels of proinflammatory cytokines like IL-6, TNFα, and IL1β, while anti-inflammatory cytokines like IL-10 remained low. Furthermore, IL-4-induced M2-like CAR-Ms could be reversed to an M1-like phenotype by PES NPs. Consistent with these results, PES NP-treated CAR-Ms exhibited increased phagocytic activity toward OC cells expressing mesothelin. Building on these in vitro results, the effect of PES NP-treated CAR-Ms on the reduction of tumor growth in OC tumor models will be further investigated. Conclusions: Our findings demonstrate that PES NPs effectively reprogram CAR-Ms to maintain a pro-inflammatory M1 phenotype. This reprogramming enhances their phagocytic activity and target cell killing in vitro. Collectively, these results support intracellular succinate delivery as a promising strategy to improve the efficacy of CAR-M-based therapies in OC. Citation Format: Remya Raja, Alexis Hand, Madhan Mohan Chandra Sekhar Jaggarapu, Jason Tonne, Richard Vile, Abhinav Acharya, Marion Curtis. Succinate based metabolic reprogramming of CAR-macrophages for ovarian cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B063.

2562 Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal cancer with limited immunotherapeutic options. Pelareorep (pela) is an intravenously delivered unmodified reovirus containing a double stranded RNA genome that has been studied as an immunotherapeutic in multiple cancers including breast, anal, colorectal and pancreatic. We previously reported high tumor response rates in first-line metastatic PDAC patients treated with pela combined with gemcitabine, nab-paclitaxel and atezolizumab. We report here the immunologic effects of pela in a cohort of first-line metastatic PDAC subjects treated with pela plus chemotherapy and atezolizumab and the correlation of these effects with tumor response. Methods: To examine its effects on pancreatic cancer, a phase 1/2 Simon 2-stage platform study (GOBLET) was performed that included patients with first-line locally advanced/metastatic unresectable PDAC. Anti-reovirus T cell activity was assessed by interferon gamma secretion (ELISPOT). Changes in the expression of plasma proteins were analyzed by Olink Response panels. T cell receptor sequencing (TCR-seq; ImmunoSEQ Assay, Adaptive Biotechnologies) was performed on tissue collected prior to the start of therapy and on blood from baseline through 3 treatment cycles to identify TILs expansion. Tumor responses were scored according to the modified RECIST v1.1 criteria. Results: Increases in anti-reovirus T cell activation as determined by ELISPOT after cycle 3 of therapy were observed in 6/8 subjects. Three subjects with maximum responses (> 300 spots) showed >30% decreases in tumor volume. Significant changes in plasma proteins as determined by Olink included PD-L1, CXCL9, CXCL10, CXCL11 and IFN-γ. Pre-treatment tumor tissue was used to identify the TIL clonal populations prior to therapy. Increased TIL clones in the blood pre-treatment was associated with tumor responses. Pela treatment increased the expansion of pre-existing and new TIL clones in the blood after one cycle of treatment. Sustained increases in pre-existing TIL clonal populations in the blood through cycle 3 of therapy were observed in subjects who exhibited reductions in tumor volume. Conclusions: These findings, while preliminary, demonstrate that pela induces not only anti-reovirus T cells but also activates innate and adaptive anti-tumor immunity in PDAC subjects treated with chemotherapy and atezolizumab. Tumor responses are associated with both the presence of TIL clones in the blood prior to treatment and the expansion of pre-existing TILs in the blood on treatment. These findings provide additional insights into the immunologic mechanisms by which pela-based therapy may provide clinical benefit in patients with metastatic PDAC.

Abstract The paradigm in the field of oncolytic virotherapy proposes that tumor cell killing by an oncolytic virus (OV) culminates in the priming of antitumor CD8 T cells. However, this ignores the impact a highly immunodominant antiviral response against the OV has on the antitumor response which has been weakened by mechanisms of central tolerance. Here, we show that inflammatory Vesicular Stomatitis Virus (VSV) failed to prime an adoptively transferred, or pre-existing, population of tumor-reactive T cells.Combination with αPD1 immune checkpoint blockade therapy improved survival only when VSV expressed tumor associated antigens (TAA). These data show that, in this model, the highly inflammatory OV VSV alone actively outcompetes antitumor immunity. However, we also show that viral expression of a mutant near-self TAA can break central tolerance expanding heteroclitic self-reactive and near-self-reactive T cells, thus overcoming viral immunodominance by promoting tumor-specific T cell proliferation in parallel with expanding antiviral T cells.

Abstract T cell immune tolerance is established in part through the activity of the Auto-immune Regulator (AIRE) transcription factor in the medullary Thymic Epithelial Cells (mTEC) of the thymus. AIRE induces expression of SELF peripheral tissue-specific antigens for presentation to naïve T cells to promote activation/deletion of potentially autoreactive T cells. We show, for the first time to our knowledge, that tumors mimic the role of AIRE in mTEC to evade immune rejection. Thus, by expressing a broad range of SELF epitopes against which minimal functional T cell reactivities exist because of thymic deletion, AIRE acts as a master controller of SELFNESS, effectively cloaking the tumor from T cell attack. Moreover, we describe a completely novel immunotherapy in which engineered changes in AIRE expression in tumor cells alters their profile of SELFNESS, exposing both AIRE-modified, and parental unmodified, tumor cells to T cell attack. Consistent with our studies, patient RNAseq shows expression of AIRE predicts response to immune therapies with a strong correlation between AIRE expression and markers of TCR signaling. Therefore, by re-setting the immunological SELFNESS of cancer cells, this novel AIRE-mediated immunotherapy 1). converts a highly tolerized T cell compartment into a heteroclitic tumor-reactive T cell population; 2) confers de novo sensitivity to immune checkpoint blockade upon non-immunogenic tumors; 3). completely removes the need to identify potentially immunogenic tumor-associated antigens as targets for generation of de novo CD8+ and helper CD4+ T cell responses; and 4) leads to potent T cell-mediated rejection of aggressive, immunologically cold, non-immunogenic tumors.

Background: Long-lived, re-activatable immunity to SARS-CoV-2 and its emerging variants will rely on T cells recognizing conserved regions of viral proteins across strains. Heterologous prime–boost regimens can elicit elevated levels of circulating CD8+ T cells that provide a reservoir of first responders upon viral infection. Although most vaccines are currently delivered intramuscularly (IM), the initial site of infection is the nasal cavity. Methods: Here, we tested the hypothesis that a heterologous prime and boost vaccine regimen delivered intranasally (IN) will generate improved immune responses locally at the site of virus infection compared to intramuscular vaccine/booster regimens. Results: In a transgenic human ACE2 murine model, both a Spike-expressing single-cycle adenovirus (SC-Ad) and an IFNß safety-enhanced replication-competent Vesicular Stomatitis Virus (VSV) platform generated anti-Spike antibody and T-cell responses that diminished with age. Although SC-Ad-Spike boosted a prime with VSV-Spike-mIFNß, SC-Ad-Spike alone induced maximal levels of IgG, IgA, and CD8+ T-cell responses. Conclusions: There were significant differences in T-cell responses in spleens compared to lungs, and the intranasal boost was significantly superior to the intramuscular boost in generating sentinel immune effectors at the site of the virus encounter in the lungs. These data show that serious consideration should be given to intranasal boosting with anti-SARS-CoV-2 vaccines.

Abstract Although immune checkpoint inhibition (ICI) has produced profound survival benefits in a broad variety of tumors, a proportion of patients do not respond. Treatment failure is in part due to immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, we developed a vesicular stomatitis virus expressing interferon-ß (VSV-IFNß) as a viro-immunotherapy against HCC. Since HCC standard of care atezolizumab/bevacizumab incorporates ICI, we tested the hypothesis that pro-inflammatory VSV-IFNß would recruit, prime, and activate anti-tumor T cells, whose activity anti-PD-L1 ICI would potentiate. However, in a partially anti-PD-L1-responsive model of HCC, addition of VSV-IFNß abolished anti-PD-L1 therapy. Cytometry by Time of Flight showed that VSV-IFNß expanded dominant anti-viral effector CD8 T cells with concomitant, relative disappearance of anti-tumor T cell populations which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, the potent anti-viral response became amalgamated with an anti-tumor T cell response generating highly significant cures compared to anti-PD-L1 ICI alone. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, by chimerizing anti-viral and anti-tumor T cell responses through encoding tumor antigens within the virus, oncolytic virotherapy can be purposed for very effective immune driven tumor clearance and can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Abstract α-PD-1 checkpoint blockade is a promising immunotherapeutic approach for many solid tumors; however, this strategy is minimally effective for GBM patients as a monotherapy. One potential reason why α-PD-1 checkpoint blockade is ineffective is the tumor microenvironment and systemic derangements of the immune system which are prevalent in GBM patients. We have successfully modeled peripheral immune derangements in experimental murine GBM models. These include low peripheral blood CD4 T cell counts, reduced MHC class II expression on monocytes, and atrophy of primary immune organs in animals harboring gliomas. We therefore hypothesized that extended half-life IL-2, a potent cytokine which promotes the proliferation, differentiation, and killing activity of T cells, could overcome immune derangements and could potentially synergize with α-PD-1 checkpoint blockade therapy. In cohorts of C57Bl/6 mice, we supplemented α-PD-1 checkpoint blockade with multiple bio-engineered extended half-life IL-2 molecules to treat GL261 and CT2A orthotopic glioma models which are normally resistant to monotherapy. We observe a statistically significant increase in survival in GL261-bearing animals receiving the combination of extended half-life IL-2 reagents and αPD-1 therapy relative to control-treated mice. Some animals receiving this combination therapy had a reduction in tumor burden to levels below detection. These long-term survivors underwent a rechallenge experiment with a second inoculation of GL261-Luc and effectively cleared tumor without further therapeutic intervention. This intervention causes a significant shift in the immune profile, including the formation of mature T cell responses, as well as restoring the diminished peripheral immune cell counts in GL261-bearing animals. Finally, combining extended half-life IL-2 with α-PD-1 checkpoint blockade is effective independently of the CD8 T cell response. Together, these data suggest that combination immunotherapies employing extended half-life IL-2 fusion proteins are translational to the clinic and do not require the identification of tumor specific antigens for efficacy

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 (EGR1) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P < 0.01; MEL624 27%, P < 0.001) and conjunctival (YUARGE 13-3064 33%, P < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

2062 Background: While cancer therapeutics have made tremendous progress within the past several decades, this benefit has not been seen in many primary cancers of the brain. Particularly confounding have been high-grade gliomas (HGG), which retain a dismal prognosis. Currently, novel therapies are being explored to rise to this unmet, critical need. One such therapy are CAR T cells, immune cells which have been engineered to target malignancy-specific antigens. Unfortunately, the efficacy of CAR T cell therapies against solid tumors is significantly limited, in large part due to impaired expansion/persistence in the immune suppressive tumor microenvironment (TME). Here we show that in vivo reactivation of CAR T cells through their native T Cell Receptor (TCR) by an oncolytic virus (OV) has therapeutic benefit in HGG. Methods: An EGFRvIII third-generation MSGV1 retroviral CAR construct containing the CD28, 4-1BB, and CD3z moieties, in tandem with the scFv derived from the human monoclonal antibody 139 and the marker Thy1.1 (38) was used to generate our CAR T cells. C57Bl/6 mice were used for in vivo experiments and both B16- and CT2A-EGFRvIII murine glioma cell lines were injected in the brain to model HGG. OVs and CAR T cells were given systemically by tail vein. OVs used include reovirus, vesicular stomatitis virus, and adenovirus. Results: By using OV in combination with EGFRvIII CAR T cells, we were able to generate a CD8 CAR population with TCR specificity for both the EGFRvIII and OV epitopes. These dual-specific (DS) CAR T expressed a memory phenotype and persisted for much longer than conventional CAR T cells. Further, we showed that these DS CAR T cells are more cytotoxic and can respond more rapidly than their conventional counterparts. We created a novel delivery mechanism for this combination OV + CAR T therapy using virus-loaded CAR T cells to bypass initial antiviral clearance from the immune system. Treatment with these OV-loaded CAR T cells lead to significant benefit in mice with HGG tumors which could be further enhanced by a systemic boost with OV, which rapidly re-activated DS CAR T cells against tumor and resulted in long-term cures of greater than 80% of treated animals. Conclusions: These promising results show that DS CAR T cells can overcome the critical therapeutic challenge of CAR T as a treatment for solid tumors. Given these promising results, we will go on to develop a clinical trial in which CAR T cells will be pre-loaded with OV and administered intravenously to patients with HGG, followed by systemic boosting with virus to re-activate DS CAR T cells against their tumor.