TRAMP-C2 Tumor Research Articles

Abstract A005: Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223

Abstract Introduction: Combination treatment with a long-acting IL-2 superkine (MDNA109FEAA-Fc; aka MDNA19) and anti-PD1 or anti-CTLA4 has shown clear evidence of synergy in blunting tumor growth. To further leverage this synergism, we designed MDNA223, a BiSKIT (Bifunctional SuperKine for ImmunoTherapy) comprising MDNA109FEAA with enhanced affinity for IL-2Rb and no binding to IL-2Ra, fused to either human or mouse anti-PD1. We present in vitro and in vivo characterization of MDNA223 and demonstrate its enhanced IL-2R agonism and effective PD1/PDL-1 blockade in multiple “hot” and “cold” syngeneic tumor models. Experimental Procedure: In vitro studies included human PBMCs and cell-based reporter assays. In vivo studies were conducted in CT26, B16F10, E0771 and TRAMP-C1 tumor models by intraperiotonneal (IP) administration. Intratumoral treatment was performed in a CT26 bilateral tumor model with or without a STING agonist. Analysis of tumor infiltrating lymphocytes (TILs) was performed using flow cytometry. Results: In studies with human PBMCs, MDNA223 showed increased p-STAT5 signaling in CD8+ T cells and reduced activity in Tregs compared to rhIL-2, consistent with its enhanced receptor selectivity. MDNA223 and anti-PD1 were similarly potent at PD1/PDL-1 blockade in a cell-based reporter assay. In mice, MDNA223 induced proliferation (based on Ki67 expression) and expansion of peripheral CD8+ T cells for over 7 days. IP treatment with MDNA223 resulted in significantly greater inhibition of CT26, B16F10, E0771 and TRAMP-C1 tumor growth when compared to combination of MDNA19 with anti-PD1 at equimolar dosage. This observation is consistent with the proposed cis-binding mechanism of MDNA223 to both, IL-2R and PD1 on the same effector immune cell. In a bilateral CT26 tumor model, IT treatment with MDNA223 not only inhibited the treated tumor but also exhibited an abscopal effect by slowing growth of the untreated tumor implanted on the opposite flank. TILs analysis of B16F10 melanoma following a single dose of MDNA223 showed increased infiltration of CD8+ T cells over Tregs compared to single agent treatment with MDNA19 or anti-PD1 as well as their combination. Treatment with MDNA223 also resulted in a larger population of functional effector CD8+ T cells expressing low levels of the Tim-3 exhaustion marker and high levels of the cytotoxic Granzyme-B protease. Conclusion: MDNA223 demonstrated superior therapeutic activity over the combination of MDNA19 and anti-PD1 in both immunologically ‘hot’ (CT26) and ‘cold’ (B16F10, E0771 and TRAMP-C1) tumor models. Additional studies with variants of MDNA223 capable of tumor selective targeting and/or conditional activation within the tumor micro-environment are in progress to maximize activity at the tumor site with tunable peripheral stimulation. Citation Format: Aanchal Sharma, Minh D To, Hardeep Kataria, Qian Liu, Rosemina Merchant, Fahar Merchant. Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A005.

Ascite-solid model of prostate cancer and its morphological characteristics

Targeted therapy and immunotherapy are considered promising novel therapies capable of increasing the effcacy of prostate cancer (PCa) treatment. The purpose of the study was to obtain and characterize TRAMP-C2 subcutaneous and ascite-solid models of prostate cancer in C57Bl/6j mice to study specifc anti-tumor activity of the candidate molecules of targeted drugs and adjust immunotherapy strategies in an evidence-based manner. Material and Methods. We used cultured TRAMP-C2 cells in subcutaneous and ascites mouse prostate cancer models. Histological and immunohistochemical methods were used to study the tumor tissues. Results. The high in vivo growth ability of TRAMP-C2 cells was demonstrated in subcutaneous and intraperitoneal inoculation of C57Bl/6j mice. These tumors are characterized by a high reproducibility and level of PSMA expression. Histological study showed that subcutaneous and carcinomatous TRAMP-C2 tumor nodules had solid structure morphologically corresponding to low differentiated neoplasm, cytomorphological analysis of smears showed that peritoneal fuid containd a large number of rounded tumor cells, macrophages and erythrocytes. Conclusion. The obtained subcutaneous and ascite-solid models of TRAMP-C2 can be useful for the development of new ways to effectively treat cancer, including targeted and immunotherapy, as well as for the experimental study of biotherapeutic effects using PSMA as a target, and photoinduced effects.

Abstract 5139: Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment

Abstract Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of malignant cells, thereby affecting tumorigenesis and tumor progression. While immune checkpoint inhibitors and agonism of costimulatory molecules can act to stimulate leukocytes, the lack of adequate recruitment of effector cells into TME often results in suboptimal immune responses. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous chemoattractant that is widely expressed by many tissues and recruits innate leukocytes through its receptor, CMKLR1 which is expressed on NK cells, some dendritic cells, and macrophages. Previous studies show that RARRES2 is downregulated across multiple tumor types including prostate cancer. Here, using a syngeneic transgenic adenocarcinoma mouse prostate (TRAMP-C1) model, we show that forced overexpression of RARRES2 by tumor cells results in significant tumor suppression and recruitment of NK and CD8+ T-cells within the TME. Male C57/BL/6 mice were inoculated with either RARRES2-overexpressing, vector control (VC) or mixed (50:50; VC: RARRES2) TRAMP-C1 cells. Chemerin overexpression in TRAMP-C1 cells did not alter their phenotypic behavior in vitro, but significantly suppressed tumor growth in vivo in mice. Furthermore, we observed a significantly higher influx of NK (CD3-CD19-NK1.1+) and CD8+ T-cells (CD3+CD8+) in tumor-infiltrating leukocytes (TILs) from mice with mixed tumors compared with VC tumors. Depletion of NK cells, CD4+, or CD8+ T cells showed that NK cells and CD8+ T-cells, but not CD4+ T-cells, were required for chemerin-dependent suppression of TRAMP-C1 tumor growth. We found significant increases in splenic NK and CD8 T cells, and a concomitant significant decrease in splenic G-MDSC in mice with chemerin-expressing tumors compared to controls, suggesting chemerin expression within the TME has an effect on systemic immune responses. Treatment of control tumors with anti-PD1 checkpoint inhibition did not result in significant tumor suppression, while anti-PD1 treatment of chemerin-expressing tumors significantly reduced tumor growth, doubling the complete response rate in these unresponsive tumors. Thus, for the first time we have demonstrated that increasing chemerin expression within the prostate TME can suppress growth by augmenting recruitment of NK and CD8+ T-cells and has the potential to be combined with checkpoint inhibitors in order to improve response rates in non-responsive tumors. Overall, these data suggest that a tumor-targeted chemerin therapeutic may have potential to treat human prostate cancers. Citation Format: Muhammad A. Saeed, Kevin Kim, Russell K. Pachynski. Chemerin suppresses prostate tumor growth by modulating the recruitment of NK and CD8+ T-cells to tumor microenvironment. [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 5139.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Onion-like doxorubicin-carrying polymeric nanomicelles with tumor acidity-sensitive dePEGylation to expose positively-charged chitosan shell for enhanced cancer chemotherapy

To effectively promote antitumor potency of doxorubicin (DOX), a regularly used chemotherapy drug, the tumor acidity-responsive polymeric nanomicelles from self-assembly of the as-synthesized amphiphilic benzoic imine-containing PEGylated chitosan-g-poly(lactic-co-glycolic acid) (PLGA) conjugates were developed as vehicles of DOX. The attained PEGylated chitosan-g-PLGA nanomicelles with high PEGylation degree (H-PEG-CSPNs) were characterized to exhibit a “onion-like” core-shell-corona structure consisting of a hydrophobic PLGA core covered by benzoic imine-rich chitosan shell and outer hydrophilic PEG corona. The DOX-carrying H-PEG-CSPNs (DOX@H-PEG-CSPNs) displayed robust colloidal stability under large-volume dilution condition and in a serum-containing aqueous solution of physiological salt concentration. Importantly, the DOX@H-PEG-CSPNs in weak acidic milieu undergoing the hydrolysis of benzoic imine bonds and increased protonation of chitosan shell showed dePEGylation and surface charge conversion. Also, the considerable swelling of protonated chitosan shell within DOX@H-PEG-CSPNs accelerated drug release. Notably, the cellular internalization of DOX@H-PEG-CSPNs by TRAMP-C1 prostate cancer cells under mimic acidic tumor microenvironment was efficiently boosted upon acidity-triggered detachment of PEG corona and exposure of positively-charged chitosan shell, thus augmenting DOX-mediated anticancer effect. Compared to free DOX molecules, the DOX@H-PEG-CSPNs appreciably suppressed TRAMP-C1 tumor growth in vivo, thereby showing great promise in improving DOX chemotherapy.

Antitumor efficacy of 90Y-NM600 targeted radionuclide therapy and PD-1 blockade is limited by regulatory T cells in murine prostate tumors

BackgroundSystemic radiation treatments that preferentially irradiate cancer cells over normal tissue, known as targeted radionuclide therapy (TRT), have shown significant potential for treating metastatic prostate cancer. Preclinical studies have demonstrated...

Abstract 4183: Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity

Abstract We sought to determine whether various inhibitors of Oxidative Phosphorylation (OxPhos) could restore tumor infiltrating T cell functionality and longevity while compromising metabolic fitness of tumor cells. Using a combination of in vitro metabolic profiling with the Seahorse XFe96 Analyzer and in vivo tumor immunotherapy in the immunocompetent TRAMP-C2 prostate adenocarcinoma model, we determined the differential impact of multiple OxPhos inhibitors on both tumor immune metabolic fitness and functional capacity. Impact on the tumor immune microenvironment of TRAMP-C2 tumors was assessed through high parameter flow cytometry on a BD X-30. While OxPhos inhibition compromised initial T cell activation, we found that two of the inhibitors increased the glucose uptake, proliferation, and activation status of effector T cells. In contrast, the same two inhibitors decreased the mitochondrial fitness, proliferation and OxPhos metabolic capacity of TRAMP-C2 tumor cells themselves. To determine whether this selective inhibition of tumor versus T cell metabolic function could improve immunotherapy responses in vivo, we implanted TRAMP-C2 tumors and treated them with these OxPhos inhibitors with or without concomitant immune checkpoint blockade (ICB). The combination of ICB and OxPhos inhibition achieved highly significant and durable rates of tumor control and regression suggesting that these approaches are potentially synergistic. Analysis of the tumor microenvironment has identified the cellular mechanisms underlying these therapeutic effects which may provide useful biomarkers as this novel combination is translated to the clinic. Citation Format: Krithikaa Rajkumar Bhanu, Priyamvada Jayaprakash, Meghan Rice, Brittany Morrow, Joseph R. Marszalek, Jason P. Gay, Christopher P. Vellano, Benjamin R. Cowen, Dean J. Welsh, Michael A. Curran. Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4183.

Hybrid PEGylated chitosan/PLGA nanoparticles designed as pH-responsive vehicles to promote intracellular drug delivery and cancer chemotherapy

To achieve effective intracellular anticancer drug release for boosted antitumor efficacy, the acidity-responsive nanovehicles for doxorubicin (DOX) delivery were fabricated by tailor-made co-assembly of amphiphilic PEGylated chitosan20k and hydrophobic poly(lactic-co-glycolic acid) (PLGA) segments at pH 8.5. The attained DOX-loaded PEGylated chitosan20k/PLGA nanoparticles (DOX-PC20kPNs) were characterized to have a spherical shape composed of drug-encapsulated chitosan20k/PLGA-constituted solid core surrounded by hydrophilic PEG shells. Compared to non-pH-sensitive DOX-loaded PLGA nanoparticles (DOX-PNs), the DOX-PC20kPNs displayed outstanding colloidal stability under serum-containing condition and tended to swell in weak acidic milieu upon increased protonation of chitosan20k within hybrid cores, thus accelerating drug release. The in vitro cellular uptake and cytotoxicity studies revealed that the DOX-PC20kPNs after being endocytosed by prostate TRAMP-C1 cancer cells rapidly liberated drug, thus promoting drug accumulation in nuclei to enhance anticancer potency. Moreover, the hydrated PEG shells of DOX-PC20kPNs remarkably reduced their uptake by macrophage-like RAW264.7 cells. Importantly, in vivo animal findings showed that the DOX-PC20kPNs exhibited the capability of inhibiting TRAMP-C1 tumor growth superior to free hydrophobic DOX molecules and DOX-PNs, demonstrating the great potential in cancer chemotherapy.

Role of Myeloid-Derived Suppressor Cells in High-Dose-Irradiated TRAMP-C1 Tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment

To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

The Role of Myeloid-derived Suppressor Cells in High-dose Irradiated TRAMP-C1 tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment

To investigate the chronological and spatial responses of myeloid-derived suppressor cells (MDSCs) to high-dose irradiation in TRAMP-C1 tumor and explore their potential role in tumor re-growth after irradiation. Murine prostate TRAMP-C1 tumors were irradiated with a single dose of 25Gy (SD-RT). Using immunohistochemical staining (IHC) and flow cytometry to verify the spatial distribution and dynamic mobilization of MDSCs. Blood and tumor lysate were collected for multiplex immunoassay. Furthermore, mice were injected Gr-1 antibody to verify the role of MDCSs in tumor re-growth after irradiation. Recruitment of Gr-1+ cells into irradiated tumors rapidly occurred in 4 hours and persisted up to 2 weeks. Flow cytometry showed that the main subpopulations of recruited MDSCs within the tumor are CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs), not CD11b+Ly6G-Ly6C- tumor-associated macrophages (TAMs). The percentage of PMN-MDSCs and M-MDSCs was also increased in peripheral blood after radiation therapy. At two weeks after irradiation, levels of multiple cytokines, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a were increased in the irradiated tumors; and levels of G-CSF, IL-6 and TNF-α were increased in the blood. The PMN-MDSCs chronologically aggregated at the central necrotic region of chronic hypoxia. The infiltrated Ly6G + PMN-MDSCs dominantly possessed pro-angiogenic factor, matrix metalloproteinase-9 (MMP-9), and highly expressed arginase-1, an immunosuppressive molecule, at the necrotic area. The depletion of MDSCs by the Gr-1 antibody had no effects on sham-irradiated tumors, but significantly delayed the regrowth of irradiated tumor. PMN-MDSCs are actively involved in tumor responses to high-dose irradiation, and their spatial distribution and phenotype changes are associated with the evolution of chronic hypoxia in the irradiated tumors. PMN-MDSCs could become a therapeutic target. Checking the MDSCs component and cytokine levels in peripheral blood could be used as an index for assessing responses in the tumor microenvironment.

Tumor microenvironment-responsive and oxygen self-sufficient oil droplet nanoparticles for enhanced photothermal/photodynamic combination therapy against hypoxic tumors

The combination of photothermal and photodynamic therapy (PTT/PDT) shows pronounced potential as a prominent therapeutic strategy for tumor treatment. However, the efficacy is limited by insufficient tumor-targeted delivery of PTT and PDT reagents and the hypoxic nature of the tumor microenvironment. To overcome these limitations, tumor acidity-responsive lipid membrane-enclosed perfluorooctyl bromide oil droplet nanoparticles (NPs) surface modified with N-acetyl histidine-modified D-α-tocopheryl polyethylene glycol 1000 succinate (PFOB@IMHNPs) were developed, capable of co-delivering oxygen, IR780 (a photothermal agent) and mTHPC (a photodynamic sensitizer) into tumors. Through self-sufficient oxygen transportation in combination with promotion of cellular uptake upon acid-triggered generation of surface positive charge, the PFOB@IMHNPs effectively delivered IR780 and mTHPC and produced singlet oxygen within hypoxic TRAMP-C1 cells following exposure to irradiation at 660 nm. This led to effective killing of hypoxic cancer cells in vitro. Importantly, when irradiation at 808 and 660 nm was carried out, PT/PD combination therapy utilizing PFOB@IMHNPs dramatically suppressed the growth of TRAMP-C1 tumors through effective tumor-targeted cargo delivery and relief of tumor hypoxia. Our results suggest the high potential of the PFOB@IMHNPs developed in this study in clinical application for cancer treatment.

Abstract 6686: SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner

Abstract SO-C101 (RLI-15) is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain designed to bypass the need of endogenous IL-15Rα, thereby leveraging the activity of IL-15 in vivo on target immune cells and reducing the toxicity of IL-15 as such. SO-C101 was previously shown to exhibit a potent anti-metastatic activity in Renca, B16F10 melanoma and delayed tumor growth in T cell-based mouse tumor models (CT26, MC38). Here we investigated the anti-tumor efficacy in predominantly natural killer (NK)-cell based mouse tumor models TC-1 and TRAMP-C2. We showed that SO-C101 monotherapy was effective in the treatment of established TC-1 tumors, which was dependent on the presence of both NK and CD8+ T cells, but not CD4+ T cells. In an early treatment setting SO-C101 significantly decreased the rate of tumor development also in dependence on NK and CD8+ T cells. SO-C101 effectively reduced tumor growth in TRAMP-C2 mice in early and advanced treatment settings. However, only in combination with anti-PD-1 antibody treatment the tumor development was prevented in majority of mice. This effect was durable, and the new tumor development was further significantly delayed after a tumor cell re-challenge, which suggests the involvement of memory T cells despite an important NK cell role in anti-tumor efficacy in these models. The efficacy of SO-C101 and anti-PD-1 treatment was not dependent on CD4+ T cells, but mainly on NK and CD8+ T cells. Interestingly, SO-C101 and anti-PD-1 treatment in double NK/CD8+ T cell-depleted mice decreased tumor growth which suggests an involvement of other immune cell populations in the anti-tumor efficacy. SO-C101 stimulated the proliferation and the cytotoxic activity of NK cells and memory CD8+ T cells without significant expansion of regulatory T cells. These data show the importance of various immune cell populations during SO-C101 monotherapy and the treatment in combination with anti-PD-1 antibodies, and set a base for further complex analysis of SO-C101 behavior. The therapeutic potential of SO-C101 is currently being tested in an ongoing Phase I clinical study in cancer patients. Citation Format: Irena Adkins, Romana Mikyskova, Nada Hradilova, Guy de Martynoff, David Bechard, Ulrich Moebius, Milan Reinis, Radek Spisek. SO-C101 displays strong anti-tumor effect in TC-1 and TRAMP-C2 tumor mice and in combination with PD-1 blockade prevents tumor development in a NK and CD8+ T cells dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6686.

IL-15 in the Combination Immunotherapy of Cancer.

We completed clinical trials of rhIL-15 by bolus, subcutaneous, and continuous intravenous infusions (CIV). IL-15 administered by CIV at 2 mcg/kg/day yielded a 38-fold increase in 10- day number of circulating NK cells, a 358-fold increase in CD56bright NK cells and a 5.8-fold increase in CD8 T cells. However, IL-15 preparations administered as monotherapy were ineffective, due to actions of immunological checkpoints and due to the lack of tumor specific targeting by NK cells. To circumvent checkpoints, trials of IL-15 in combination with other anticancer agents were initiated. Tumor-bearing mice receiving IL-15 with antibodies to CTLA-4 and PD-L1 manifested marked prolongation of survival compared to mice receiving IL-15 with either agent alone. In translation, a phase I trial was initiated involving IL-15 (rhIL-15), nivolumab and ipilimumab in patients with malignancy (NCT03388632). In rhesus macaques CIV IL-15 at 20 μg/kg/day for 10 days led to an 80-fold increase in number of circulating effector memory CD8 T cells. However, administration of γc cytokines such as IL-15 led to paralysis/depression of CD4 T-cells that was mediated through transient expression of SOCS3 that inhibited the STAT5 signaling pathway. This lost CD4 helper role could be restored alternatively by CD40 agonists. In the TRAMP-C2 prostate tumor model the combination of IL-15 with agonistic anti-CD40 produced additive effects in terms of numbers of TRAMP-C2 tumor specific Spas/SCNC/9H tetramer positive CD8 T cells expressed and tumor responses. A clinical trial is being initiated for patients with cancer using an intralesional anti-CD40 in combination with CIV rhIL-15. To translate IL-15-mediated increases in NK cells, we investigated combination therapy of IL-15 with anticancer monoclonal antibodies including rituximab in mouse models of EL-4 lymphoma transfected with human CD20 and with alemtuzumab (CAMPATH-1H) in a xenograft model of adult T cell leukemia (ATL). IL-15 enhanced the ADCC and therapeutic efficacy of both antibodies. These results provided the scientific basis for trials of IL-15 combined with alemtuzumab (anti-CD52) for patients with ATL (NCT02689453), with obinutuzumab (anti-CD20) for patients with CLL (NCT03759184), and with avelumab (anti-PD-L1) in patients with T-cell lymphoma (NCT03905135) and renal cancer (NCT04150562). In the first trial, there was elimination of circulating ATL and CLL leukemic cells in select patients.

Sunitinib Treatment-elicited Distinct Tumor Microenvironment Dramatically Compensated the Reduction of Myeloid-derived Suppressor Cells.

The clinical response rate of prostate cancer to tyrosine kinase inhibitor (TKI) monotherapy is low. The mechanisms of resistance to TKI are unclear. This study aimed to examine if the tumor microenvironment (TME) is involved in the resistance. The anti-vascular effect of Sutent was examined by immunofluorescent staining in TRAMP-C1 tumor. The percentage of CD11b+ population were analyzed by flow cytometry. The level of cytokines and chemokines were measured by multiplex immunoassay. The Sutent monotherapy caused 1.5 days of tumor growth delay, chronic hypoxia, and more mature vasculature. Sutent monotherapy increased the percentage of polymorphonuclear myeloid-derived suppressor cells (MDSCs) in peripheral blood. The evolved TME triggered the re-distribution of myeloid cells in chronically hypoxic areas. The multiplex immunoassay indicated higher levels of several cytokines and chemokines both in tumors and the blood. Sunitinib treatment induced a distinct tumor microenvironment that impaired the efficient reduction of MDSCs by TKI.

Abstract 3960: Low doses of Docetaxel in combination with anti-prostate cancer immunotherapy lead to complete tumor-free outcome through Galectin-3 silencing

Abstract Based on animal model results and the analysis of clinical data from metastatic and castration resistant prostate cancer (mCRPC) patients, we were able to demonstrate the advantages and the mechanisms by which Docetaxel(DTX)-based chemotherapy induces the correct pre-conditioning of the tumour microenvironment to allow high cytotoxic T cell activity to avoid prostate tumour growth and recurrence. First, analysis of the gene expression of human PCa cells showed that treatment with DTX decreases the expression of Galectin-3 (Gal-3) in either chemo-sensitive or -resistant prostate cancer (PCa) cell lines. In addition, using a murine PCa model (derived from TRAMP mice), treatment of TRAMP-C1 (TC1) cells or tumors with a low dose of DTX lowered the expression of Gal-3 but not Gal-1 (among others) in this cell line and tumors as well (reversible silencing of about 98% (in vitro) and 55% (in vivo)). More importantly, Gal-3 downregulation was confirmed in clinical samples of mCRPC patients treated by taxane-based chemotherapy. To evaluate whether this DTX-dependent Gal-3 downregulation in PCa tumors could be one of the principal molecular factors responsible for chemotherapy enhancement of immunotherapy, we decide to inoculate mice with an autologous BM-DC vaccine loaded with lysates from TC1 cells pretreated by DTX in vitro or expressing an anti-Gal-3 shRNA. This protocol completely protects the animals to the growth of Gal-3 deficient-PCa tumors. We then decided to go further in the understanding of the mechanisms that promote this effective anti-PCa immune response, and analysed the proliferation ability of lymphocytes in the presence or absence of TC1 tumor cells, expressing or not Gal-3. The results were encouraging as we demonstrated that Gal-3 down-regulation in tumors (through the use of TC1-shGal-3 cells or TC1-treated with low doses of DTX) was sufficient to restore the capability of CD8+ T cells to be activated and to proliferate as well as in the absence of tumor cells. Interestingly, we showed by ex vivo cytotoxicity assays that these activated and proliferating CD8+ T lymphocytes after vaccination of mice with TC1-lysate loaded BM-DC are capable of killing Gal-3 downregulated TC1 cells and protect prostate cancer recurrence and metastasis development in mice after primary tumor-resection. In conclusion, our results strongly suggest that Gal-3 is one of the principal immunosuppressor in prostate cancer that limits the success of immunotherapy. We then show that DTX-based chemotherapy would promote a decrease of Gal-3 expression by the tumors to create a permissive tumor microenvironment for successful immunotherapy. Finally, we propose a rational protocol combining no toxic/low doses of Docetaxel with simplified immunotherapy to completely control tumor recurrence for all PCa patients and chemoresistant as well. Citation Format: Daniel Compagno, Carolina Tiraboschi, Lucas Gentilini, Enrique Corapi, Felipe M. Jaworski, Anne Chauchereau, Diego J. Laderach. Low doses of Docetaxel in combination with anti-prostate cancer immunotherapy lead to complete tumor-free outcome through Galectin-3 silencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3960.

Preclinical Development of a WT1 Oral Cancer Vaccine Using a Bacterial Vector to Treat Castration-Resistant Prostate Cancer

Previously, we constructed a recombinant Bifidobacterium longum displaying a partial mouse Wilms' tumor 1 (WT1) protein (B. longum 420) as an oral cancer vaccine using a bacterial vector and demonstrated that oral administration of B. longum 420 significantly inhibited tumor growth compared with the Db126 WT1 peptide vaccine in the TRAMP-C2, mouse castration-resistant prostate cancer (CRPC) syngeneic tumor model. The present study demonstrated that oral administration of 1.0×109 colony-forming units of B. longum 420 induced significantly higher cytotoxicity against TRAMP-C2 cells than intraperitoneal injection of 100 μg of Db126, and the in vivo antitumor activity of B. longum 420 in the TRAMP-C2 tumor model could be augmented by intraperitoneal injections of 250 μg of anti-PD-1 antibody. For the clinical development, we produced the B440 pharmaceutical formulation, which is lyophilized powder of inactivated B. longum 440 displaying the partially modified human WT1 protein. We confirmed that B. longum 440 could induce cellular immunity specific to multiple WT1 epitopes. In a preclinical dosage study, B440 significantly inhibited growth of the TRAMP-C2 tumors compared with that of the control groups (PBS and B. longum not expressing WT1) at all dosages (1, 5, and 10 mg/body of B440). These mouse doses were considered to correspond with practical oral administration doses of 0.2, 1, and 2 g/body for humans. Taken together, these results suggest that the B440 WT1 oral cancer vaccine can be developed as a novel oral immuno-oncology drug to treat CRPC as a monotherapy or as an adjunct to immune checkpoint inhibitors.

Abstract B189: Tumoral STING is required for effective anticancer immunity

Abstract Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that catalyses the synthesis of cGAMP, which serves as a ligand for stimulator of interferon (IFN) genes (STING). Activation of STING results in production of type I IFNs through phosphorylation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). Type I IFNs are critical participants in the innate and adaptive immune recognition of cancer cells. Deficiencies in the cGAS-STING signalling pathway have been reported in many tumors. This mitigates expression of type I IFNs and may thus contribute to non-inflamed tumor microenvironment. In particular, a non-T-cell-inflamed tumor microenvironment correlates with poor patient survival. STING agonists may contribute to antitumor activity by upregulating proinflammatory cytokines and type I IFNs and various STING agonists are now being tested in clinical trials. The role of STING in immune cells is relatively well understood; however, its role in tumor cells has not yet been described in detail. Here we show that cGAS is able to bind to DNA present in the cytosol of tumor cells and subsequently induces STING signaling leading to expression of type I IFNs. Knockout of STING in mouse prostate TRAMP-C2 tumor cells resulted in higher tumor burden and reduced infiltration of immune cells such as dendritic and CD8+ T-cells into the tumor microenvironment. Consistently, treatment with the STING agonist cGAMP elevated type I IFNs levels in TRAMP-C2 cells and led to a reduced tumor volume compared to untreated control. This suggests a pivotal role of tumoral STING in antitumor immunity. However, despite intact STING expression, most tested human cancer cell lines were not responsive to various STING agonists and consequently failed to upregulate expression of type I IFNs. In contrast, all tested tumor cell lines responded to Poly(I:C)-induced TLR3 signaling, suggesting that failure to respond to cGAMP was due to a defect upstream of TBK1. Downregulation of cGAS did not render cells responsive to cGAMP, indicating that inability to respond to cGAMP is due to deficiencies of STING to activate TBK1. In the human and mouse prostate cancer cell lines DU145 and TRAMP-C2, respectively, autocrine IL-6 rendered cells unresponsive to STING agonists. While treatment with anti-IL-6 antibodies restored cGAMP responsiveness in DU145 cells, addition of recombinant IL-6 suppressed cGAMP-mediated upregulation of type I IFNs. In summary, our data suggest that cytosolic DNA activates the cGAS-STING signaling pathway. A functional STING is pivotal for eliciting an effective anticancer immune response. In most human cancer cell lines, however, STING signalling is inhibited. Since STING agonists are being evaluated in clinical trials, it is crucial to understand mechanisms that mediate STING unresponsiveness. We show that in tested prostate cancer cells, IL-6 signals contribute to unresponsiveness of STING and blocking of IL-6 can restore responsiveness towards STING agonists. Citation Format: Manuel Adrian Suter, Wendy Ya Ling Zhang, Muznah Bte Nazar Khan Khatoo, Nikki Ya Ling Tan, Chien Tei Too, Shubhita Tripathi, Paul A. MacAry, Veronique Angeli, Stephan Gasser. Tumoral STING is required for effective anticancer immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B189.

A role for paracrine interleukin-6 signaling in the tumor microenvironment in prostate tumor growth.

Interleukin-6 (IL-6) is a mediator of inflammation that can facilitate prostate cancer progression. We previously demonstrated that IL-6 is present in the prostate tumor microenvironment and is restricted almost exclusively to the stromal compartment. The present study examined the influence of paracrine IL-6 signaling on prostate tumor growth using allograft models of mouse prostate cancer (TRAMP-C2), colon cancer (MC38), and melanoma (B16) cell lines in wildtype (WT) and IL-6 knockout (IL-6-/- ) mice. Cells were implanted into WT or IL-6-/- mice and tumor sizes were measured at a 3 to 4 day interval. Serum, tumors, and other organs were collected for IL-6 analysis by ELISA and RNA in situ hybridization (RISH). There was a significant reduction in TRAMP-C2 and B16 tumor size grown in IL-6-/- mice versus WT mice (P = 0.0006 and P = 0.02, respectively). This trend was not observed for the MC38 cell line. RISH analysis of TRAMP-C2 tumors grown in WT mice showed that cells present in the tumor microenvironment were the primary source of IL-6 mRNA, not the TRAMP-C2 cells. Serum IL-6 ELISA analyses showed an increase in the circulating levels of IL-6 in WT mice bearing TRAMP-C2 tumors. Similar phospho-STAT3 expression and tumor vascularization were observed in TRAMP-C2 tumors grown in WT and IL-6-/- mice. Our results are consistent with previous studies in prostate cancer patients demonstrating that paracrine IL-6 production in the tumor microenvironment may influence tumor growth. Additionally, these data provide evidence that elevated systemic IL-6 levels may be involved in tumor growth regulation in prostate cancer, and are not simply caused by or indicative of tumor burden.

Omega-3 fatty acids decrease prostate cancer progression associated with an anti-tumor immune response in eugonadal and castrated mice.

Several lines of evidence suggest effects of dietary fat on prostate cancer (PCa) development and progression. Targeting omega (ω)-3:ω6 fatty acids (FA) ratio could be beneficial against PCa by favorably modulating inflammation. Here, we studied the effects of ω3- and ω6-enriched diets on prostate tumor growth and inflammatory response in androgen-deprived and non-deprived conditions. Immune-competent eugonadal and castrated C57BL/6 mice were injected with TRAMP-C2 prostate tumor cells and daily fed with ω3- or ω6-enriched diet. FA and cytokine profiles were measured in blood and tumors using gas chromatography and multiplex immunoassay, respectively. Immune cell infiltration in tumors was profiled by multicolor flow cytometry. ω3-enriched diet decreased prostate TRAMP-C2 tumor growth in immune-competent eugonadal and castrated mice. Cytokines associated with Th1 immune response (IL-12 [p70], IFN-γ, GM-CSF) and eosinophil recruitment (eotaxin-1, IL-5, and IL-13) were significantly elevated in tumors of ω3-fed mice. Using in vitro experiments, we confirmed ω3 FA-induced eotaxin-1 secretion by tumor cells and that eotaxin-1 secretion was regulated by androgens. Analysis of immune cell infiltrating tumors showed no major difference of immune cells' abundance between ω3- and ω6-enriched diets. ω3-enriched diet reduces prostate tumor growth independently of androgen levels. ω3 FA can inhibit tumor cell growth and induce a local anti-tumor inflammatory response. These findings warrant further examination of dietary ω3's potential to slow down the progression of androgen-sensitive and castrate-resistant PCa by modulating immune cell function in tumors.

Abstract 3849: Identification and optimization of chemical compounds as potent agonists of human STING with anticancer activity in mice

Abstract The host STING pathway plays a critical role in innate immune sensing of cancer, that drives type-I interferons (IFNs) production and promotes aggressive antitumor responses. Compound DMXAA is an agonist of mouse STING (mSTING) and demonstrated potent antitumor activities in several tumor models, including melanoma. However, DMXAA cannot activate human STING (hSTING), which provides a possible rationale for its failure in recent clinical trial. In this project, we aim to identify novel chemical compounds as potent STING agonists. By combining structure-based drug design and in vitro assays, we have discovered two initial hits as STING agonists that belong to two different chemical scaffolds. Direct binding of our compounds with hSTING was confirmed by Surface plasmon resonance (SPR) analysis. Chemical optimization of our initial hits has led to compound #150 and #171. We demonstrated that #150 and #171 at 10 uM have substantially activated STING pathway in HEK293 cells that were transiently transfected with plasmid expressing hSTING. Compound #171 at 20 uM potently activated STING signaling in human THP-1 cells that harbor hSTINGHAQ as well as PBMC cells from a panel of human donors. Importantly, with DMXAA as the control, we demonstrated that #171 at 20 mg/kg via i.p. has significant antitumor effect against TRAMP-c1 tumor in C57/BL6 mice. Citation Format: Jian Hui Wu. Identification and optimization of chemical compounds as potent agonists of human STING with anticancer activity in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3849.