Cell-dependent Anti-tumor Response Research Articles

Chemical Modifications to Enhance the Drug Properties of a VIP Receptor Antagonist (ANT) Peptide.

Antagonist peptides (ANTs) of vasoactive intestinal polypeptide receptors (VIP-Rs) are shown to enhance T cell activation and proliferation in vitro, as well as improving T cell-dependent anti-tumor response in acute myeloid leukemia (AML) murine models. However, peptide therapeutics often suffer from poor metabolic stability and exhibit a short half-life/fast elimination in vivo. In this study, we describe efforts to enhance the drug properties of ANTs via chemical modifications. The lead antagonist (ANT308) is derivatized with the following modifications: N-terminus acetylation, peptide stapling, and PEGylation. Acetylated ANT308 exhibits diminished T cell activation in vitro, indicating that N-terminus conservation is critical for antagonist activity. The replacement of residues 13 and 17 with cysteine to accommodate a chemical staple results in diminished survival using the modified peptide to treat mice with AML. However, the incorporation of the constraint increases survival and reduces tumor burden relative to its unstapled counterpart. Notably, PEGylation has a significant positive effect, with fewer doses of PEGylated ANT308 needed to achieve comparable overall survival and tumor burden in leukemic mice dosed with the parenteral ANT308 peptide, suggesting that polyethylene glycol (PEG) incorporation enhances longevity, and thus the antagonist activity of ANT308.

Abstract IA022: Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors

Abstract Most chemotherapies target DNA replication, and their efficacy depends on the DNA damage response that integrates DNA repair with the cell cycle. Widely used standard-of-care antimetabolites inhibit thymidylate synthase and increase the incorporation of deoxyuridine (dU) into DNA by polymerases. Contamination of dU in DNA is limited by the DNA damage response kinase ATR that induces cell cycle checkpoints and reduces the rate of DNA replication. ATR inhibitors (ATRi) induce origin firing across active replicons and cause ribonuclease reductase degradation in otherwise unperturbed cells. This increases both the amount of DNA replication and the amount of free dUTP in cells. Thus, ATRi increase the incorporation of dU into DNA by polymerases. Since ATRi also inhibit cell cycle checkpoints, ATRi induce more dU contamination than antimetabolites. ATRi-induced dU contamination in DNA is associated with an innate immune response. We showed this with the simple observation that ATRi-induced dU contamination and IFN-α/β expression is reversed by low doses of thymidine. Here we show that ATRi-induced IFN-α/β response is dependent on uracil DNA glycosylase (UNG) which removes dU from DNA. Our data are consistent with a model in which UNG-dependent base excision repair (BER) removes dU from DNA, ultimately generating cytoplasmic dsDNA that induces IFN-α/β. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8+ T cell dependent anti-tumor responses in mouse models of cancer treated with radiation therapy. Citation Format: Pinakin Pandya, Frank P. Vendetti, Joseph A. Ghoubaira, Sudipta Pathak, Joshua J. Deppas, Reyna E. Jones, Yunqi Zhang, Daniel Ivanov, Raquel Buj, Katherine M. Aird, Jan H. Beumer, Robert W. Sobol, Christopher J. Bakkenist. Uracil DNA glycosylase activity limits deoxyuridine contamination in genomic DNA and is essential for the type-1 interferon response in cells treated with ATR kinase inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA022.

Tumor cell–derived spermidine is an oncometabolite that suppresses TCR clustering for intratumoral CD8+ T cell activation

The activation and expansion of T cells that recognize cancer cells is an essential aspect to antitumor immunity. Tumors may escape destruction by the immune system through ectopic expression of inhibitory immune ligands typically exemplified by the PD-L1/PD-1 pathway. Here, we reveal another facet of tumor evasion from T cell surveillance. By secretome profiling of necrotic tumor cells, we identified an oncometabolite spermidine as a unique inhibitor of T cell receptor (TCR) signaling. Mechanistically, spermidine causes the downregulation of the plasma membrane cholesterol levels, resulting in the suppression of TCR clustering. Using syngeneic mouse models, we show that spermidine is abundantly detected in the tumor immune microenvironment (TIME) and that administration of the polyamine synthesis inhibitor effectively enhanced CD8+ T cell-dependent antitumor responses. Further, the combination of the polyamine synthesis inhibitor with anti-PD-1 immune checkpoint antibody resulted in a much stronger antitumor immune response. This study reveals an aspect of immunosuppressive TIME, wherein spermidine functions as a metabolic T cell checkpoint that may offer a unique approach for promoting tumor immunotherapy.

The schedule of ATR inhibitor AZD6738 can potentiate or abolish antitumor immune responses to radiotherapy.

Inhibitors of the DNA damage signaling kinase ATR increase tumor cell killing by chemotherapies that target DNA replication forks but also kill rapidly proliferating immune cells including activated T cells. Nevertheless, ATR inhibitor (ATRi) and radiotherapy (RT) can be combined to generate CD8+ T cell-dependent antitumor responses in mouse models. To determine the optimal schedule of ATRi and RT, we determined the impact of short-course versus prolonged daily treatment with AZD6738 (ATRi) on responses to RT (days 1-2). Short-course ATRi (days 1-3) plus RT caused expansion of tumor antigen-specific, effector CD8+ T cells in the tumor-draining lymph node (DLN) at 1 week after RT. This was preceded by acute decreases in proliferating tumor-infiltrating and peripheral T cells and a rapid proliferative rebound after ATRi cessation, increased inflammatory signaling (IFN-β, chemokines, particularly CXCL10) in tumors, and an accumulation of inflammatory cells in the DLN. In contrast, prolonged ATRi (days 1-9) prevented the expansion of tumor antigen-specific, effector CD8+ T cells in the DLN, and entirely abolished the therapeutic benefit of short-course ATRi with RT and anti-PD-L1. Our data argue that ATRi cessation is essential to allow CD8+ T cell responses to both RT and immune checkpoint inhibitors.

Abstract IA016: Determinants of B cell fate and function in cancer

Abstract Effector B cell responses in solid malignancies are associated with anti-tumor immunity and favorable response to immunotherapy. B cells can amplify anti-tumor immune responses through antibody production, antigen presentation, and pro-inflammatory cytokine release; yet B cells in cancer patients and tumor-bearing mice often fail to support these effector cell functions. The determinants of B cell fate and function in cancer are still not clear. We find that systemic exposure to STING agonists potentiates expansion of immunosuppressive regulatory human and mouse IL-35+ B lymphocytes in an IRF3 dependent manner. Mechanistically, the loss of STING signaling in B cells or treatment with cGAMP combined with ablation of IL-35 facilitates increases NK cell proliferation and enhances NK cell dependent anti-tumor response. Further, we identify dysregulated transcriptional programs activated in pancreatic cancer-associated B cells that promote differentiation of naïve into immunosuppressive B cells and inhibit differentiation of anti-tumor plasma cells. The signaling network that contributes to this dysfunction is driven by IL35-mediated formation of STAT3-PAX5 complex that upregulates the transcriptional factor BCL6 in B cells. Transient inhibition of BCL6 in naïve B cells is sufficient to stimulate the intratumoral accumulation of plasma cells and effector T cells and renders pancreatic tumors sensitive to anti-PD-1 immune checkpoint blockade therapy. Our findings reveal that systemic STING signaling can expand immunosuppressive B cells and show that B cell differentiation can be re-routed towards anti-tumor plasma cell response, suggesting novel combinatorial strategies to facilitate anti-tumor immune activity. Citation Format: Yuliya Pylayeva-Gupta. Determinants of B cell fate and function in cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA016.

352 (PB132) - Subasumstat, a first-in-class inhibitor of SUMO-activating enzyme, demonstrates dose-dependent target engagement and SUMOylation inhibition, leading to rapid activation of innate and adaptive immune responses in the dose escalation portion of a phase 1/2 clinical study

Background: Subasumstat (TAK-981), a small-molecule inhibitor of SUMOylation, promotes a type-1 interferon (IFN-1) response, leading to activation of the immune system with the potential to augment antitumor immunity. Previous preclinical data suggest that subasumstat enhances antigen cross-presentation and promotes T and NK cell-dependent antitumor responses in syngeneic mouse models. Here, we report pharmacodynamic (PD) data from the dose-escalation portion of a phase 1/2 clinical study (NCT03648372).

Abstract 4155: Mitazalimab, a potent CD40 agonist in combination with chemotherapy redirects and activates tumor infiltrating myeloid cells

Abstract Mitazalimab is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on myeloid cells, such as dendritic cells and tumor infiltrating macrophages, leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. Mitazalimab is a FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and manageable safety profile. Immunologically cold and immune-excluded tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, effector CD8+ T cells are excluded from the tumor by the desmoplastic tumor stroma, which surrounds the tumor and hosts immunosuppressive macrophages that dampen the immune response in the tumor microenvironment. By activating and re-directing tumor infiltrating myeloid cells, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective anti-tumor response by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor thereby enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy, inducing long-term survival. The combined treatment improved activation of antigen presenting cells in the circulation, intratumoral T cell responses as well as reduced the amount of intratumoral immunosuppressive M2 macrophages. By converting the MB49 tumor cell line resistant to FOLFIRINOX treatment, we could further demonstrate that the combination of mitazalimab and FOLFIRINOX induced a strong anti-tumor activity also in a chemoresistant variant of the MB49 cell line. In conclusion, mitazalimab synergizes effectively with chemotherapy, leading to induction of long-term survival in a preclinical tumor model by reducing immunosuppressive M2 macrophages and improving T cell responses intratumorally. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099), where mitazalimab was well tolerated up to 1200 μg/kg with a manageable safety profile, support the ongoing clinical phase 2 study OPTIMIZE-1 (NCT04888312) of mitazalimab in combination with chemotherapy in pancreatic cancer. Citation Format: Karin Enell Smith, Mia Thagesson, Anneli Nilsson, Doreen Werchau, Peter Ellmark. Mitazalimab, a potent CD40 agonist in combination with chemotherapy redirects and activates tumor infiltrating myeloid cells [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 4155.

A phase 1b, multicenter, dose-escalation study of subasumstat (TAK-981) in combination with pembrolizumab in patients (pts) with advanced solid tumors.

2506 Background: SUMOylation is a post-translational modification with a role in limiting type 1 interferon (IFN-1)-dependent immune responses. Subasumstat is a small-molecule inhibitor of SUMOylation with the potential to increase antitumor immunity and overcome tumor resistance to checkpoint inhibitors (CPI) by inducing IFN-1 signaling. Preclinical data suggest that subasumstat enhances antigen cross-presentation, promoting T cell dependent antitumor responses; subasumstat plus an anti-PD-1 CPI has shown synergistic tumor growth inhibition and activation of CD8+ T cells and natural killer cells in syngeneic mouse models. We report data from the dose escalation part of a phase 1b study of subasumstat with pembrolizumab in pts with relapsed/refractory, CPI-exposed, non-squamous non-small-cell lung cancer (NSCLC) or microsatellite-stable colorectal cancer (MSS-CRC). Methods: Pts received escalating doses (40, 60, 90, and 120 mg) of subasumstat IV in 3 dosing schedules: days 1 and 8 (QW), days 1, 4, 8, and 11 (BIW), or days 1, 8, and 15 (90 mg only) of 21-day cycles, plus pembrolizumab 200 mg IV on day 1 of each cycle for 2 years or until disease progression or unacceptable toxicity. Dose escalation was guided by Bayesian optimal interval design. Phase 1b primary objectives were safety, tolerability, and the recommended phase 2 dose of subasumstat with pembrolizumab. Results: As of October 13, 2021, 43 (35 MSS-CRC; 8 NSCLC) pts had received ≥1 dose of subasumstat (22 BIW [40–120 mg]; 15 QW [90 –120 mg]; 6 on days 1, 8, 15 [90 mg)]. Median number of treatment cycles was 3; 28 (65%) pts had discontinued treatment, 21 (49%) due to progressive disease. Median age was 58 years (range 34–77); 56% of pts were male. One pt had a dose-limiting toxicity of grade 3 angioedema at 120 mg BIW. The maximum tolerated dose was not identified. Treatment-emergent adverse events (TEAEs) occurred in 38 (88%) pts; subasumstat-related TEAEs occurred in 34 (79%) pts, and included chills in 20 (47%), pyrexia in 16 (37%), fatigue in 9 (21%), anemia in 6 (14%), and stomatitis in 5 pts (12%; 3 [50%] in the 120 mg BIW cohort). Grade ≥3 TEAEs occurred in 24 (56%) pts; subasumstat-related grade ≥3 TEAEs reported in 10 (23%) pts included anemia, pyrexia, and increased aspartate aminotransferase (2 [5%] pts each). Pharmacokinetic activity of subasumstat was linear and decreased in a tri-phasic manner. Subasumstat exerted pharmacodynamic activity including target engagement, SUMOylation inhibition and increased IFN-1 signaling. Partial responses were observed at ≥40 mg dose levels in pts with NSCLC and MSS-CRC. Conclusions: Subasumstat plus pembrolizumab showed a favorable safety profile and promising anti-tumor activity in pre-treated NSCLC and MSS-CRC pts. Updated data will be presented at the meeting. Subasumstat plus pembrolizumab is currently in phase 2 clinical development (NCT04381650). Clinical trial information: NCT04381650.

Stapled Liposomes Enhance Cross‐Priming of Radio‐Immunotherapy

The release of tumor-associated antigens (TAAs) and their cross-presentation in dendritic cells (DCs) are crucial for radio-immunotherapy. However, the irradiation resistance of tumor cells usually results in limited TAA generation and release. Importantly, TAAs internalized by DCs are easily degraded in lysosomes, resulting in unsatisfactory extent of TAA cross-presentation. Herein, an antigen-capturing stapled liposome (ACSL) with a robust structure and bioactive surface is developed. The ACSLs capture and transport TAAs from lysosomes to the cytoplasm in DCs, thereby enhancing TAA cross-presentation. l-arginine encapsulated in ACSLs induces robust T cell-dependent antitumor response and immune memory in 4T1 tumor-bearing mice after local irradiation, resulting in significant tumor suppression and an abscopal effect. Replacing l-arginine with radiosensitizers, photosensitizers, and photothermal agents may make ACSL a universal platform for the rapid development of various combinations of anticancer therapies.

Abstract PO-072: Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-072.

Inhibition of Vasoactive Intestinal Peptide Signaling with More Potent Inhibitors Augments T-Cell Activation and Prolongs Survival in Leukemic Mice

Introduction: Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide with immunosuppressive effects on T cells. Inhibition of VIP receptor (VIP-R) signaling by VIPhyb, a first-generation VIP-R antagonist, not only enhances T-cell activation and proliferation in vitro but also improves T cell dependent anti-tumor response in mouse models of acute myeloid leukemia (AML) and T lymphoblastic leukemia (Li et al. 2016; Petersen, Li, and Waller 2017). The goal of the project is to develop more potent VIP-R antagonists that generate a significantly more robust anti-tumor response in mouse models of AML, when compared to VIPhyb and validate a screening method to test the efficacy of novel peptides in activating human T cells in vitro. In this study, we report, for the first time, the activity of novel VIP-R antagonists on the activation profile of human T cells.Methods: We utilized in-silico-based modeling to identify 10 novel VIP-R antagonists from a library of 300 peptide sequences predicted to have increased binding affinity to VIP receptors VPAC1 and VPAC2 when compared to VIP or VIPhyb (Table 1). The library was generated from peptide sequences that contain the six charged N-terminal residues of the neurotensin present in VIPhyb with two or more amino acid substitutions within the C-terminal amino acid sequence of VIP. The ability of these peptides was tested in vitro using T cells from multiple healthy human donors activated using anti-CD3 monoclonal antibody coated plates. Activation status was assessed by flow cytometry of CD69, OX40, PD1, Tim3 and Lag3 expression relative to control cultures without added peptides. Potency of the novel antagonists in vivo was tested in a mouse AML model, by treating C1498- bearing mice with subcutaneous administration of VIP, VIPhyb, scrambled peptide (SCRAM1) or the second-generation VIP-R antagonists (labeled as ‘ANT‘) from day 6-12 after tumor implantation.Results: Inhibiting VIP-R signaling in human T cells using second-generation VIP-R antagonists ANT008, ANT308 and ANT195 showed approximately 1.5-to-2-fold increase in CD69, OX40, Tim3, Lag3 and OX40 expression in CD4+ T cells following 24-hour of drug exposure compared to control cultures (Figure 1A). A smaller effect of VIP-R antagonists on activation of CD8+ subsets was observed (Figure 1B). Among the peptides, ANT195 was superior to ANT008 and ANT308 which shows potency even at 1μM compared to 3μM for ANT008 and ANT308. However, significant increase in CD69 expression was observed in both CD4+ and CD8+ T cells in cultures treated with ANT308 (Figure 1 A&B, *p<0.05). Viability of the T cells was not affected by incubation with the queried peptides (Data not shown). These data corresponded to in vivo activity of the novel VIP-R antagonists such as ANT308 and ANT195 which rendered 40% of mice leukemia-free at day 60 compared to only 5% long-term survival with VIPhyb (Figure 2). Another candidate, ANT300, increased median survival time (MST) by up to 47 days compared to MST of 34 days with VIPhyb (Figure 2).Conclusions: Here, we report a simple and robust in vitro method to screen for immune activity potential of novel second-generation VIP-R antagonists using human T cells. Preliminary screen shows VIP-R antagonists augment activation of both CD4+ and CD8+ T cells. Our results indicate that ANT308 and ANT195 are more potent VIP-R antagonists with enhanced activity in vitro (human) and in vivo (mouse) than VIPhyb and ANT008, which demonstrate lower predicted binding affinities to VPAC1 and VPAC2. Our study supports the hypothesis that higher predicted binding affinity to VPAC1 and/or VPAC2 is associated with enhanced activity in stimulating human T cells and promoting anti-leukemia activity in mice. Further mechanistic studies on how inhibition of VIP-R signaling augments T cell activation and function are underway. These novel antagonists can lead to peptide-based immunotherapy for the treatment of various liquid cancers. Clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies. [Display omitted] DisclosuresWaller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

Bi-specific macrophage nano-engager for cancer immunotherapy

Immunotherapy holds great promise to improve the cancer treatment. The uptake of tumor-associated antigens (TAAs) by tumor-infiltrating antigen-presenting cells (e.g., macrophages) is the essential step for achieving efficient antitumor immunity. However, tumor cells usually evade phagocytosis of macrophage, resulting in inefficient TAA uptake. Herein, we demonstrate a bi-specific macrophage nano-engager (BiME) that can enhance the recognition and phagocytosis of tumor cells by macrophages, thereby achieving effective TAA uptake and presentation. BiME is composed of an albumin-based nanoparticle with a surface of crosslinked polymer network containing tumor-targeting moieties, macrophage-targeting moieties, and detachable PEG. Upon entering tumor tissues, BiME detaches the PEG and triggers phagocytosis of tumor cells by macrophages. As a result, BiME converts the tumor cells into an in situ vaccine, thereby activating robust T cell-dependent antitumor responses. By changing the tumor-targeting moieties, BiME may become a universal strategy to enhance the antitumor immunity against a broad range of solid tumors.

Abstract 1593: Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy

Abstract Mitazalimab (ADC-1013, JNJ-64457107) is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on antigen-presenting cells (APC), leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. There are several CD40 agonists in clinical development with different characteristics, affecting both efficacy and tolerability. Mitazalimab is an FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and safety. Immunologically “cold” tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, infiltration of effector CD8+ T cells is blocked by the desmoplastic tumor stroma, which surrounds the tumor and hosts immune-suppressive macrophages that dampen the immune response in the tumor microenvironment (TME). By activating and re-directing the immunosuppressive M2 macrophages into tumoricidal M1 macrophages in the TME, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective immune reaction by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy and induced a long-term survival. Further, it was also demonstrated that mitazalimab induced a concentration-dependent activation of tumor-associated macrophages (TAMs), purified from human tumor samples, into a more tumoricidal M1 like phenotype, by upregulation of cell surface markers such as CD83.In conclusion, mitazalimab re-directs human macrophages into a M1 like tumoricidal, less immunosuppressive phenotype, and synergizes effectively with chemotherapy, leading to induction of a long-term survival in a preclinical tumor mouse model. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099) where mitazalimab was well tolerated up to 1200 μg/kg with manageable side effects, support further clinical development of mitazalimab in combination with chemotherapy in pancreatic cancer. Citation Format: Adnan Deronic, Mia Thagesson, Anneli Nilsson, Peter Ellmark, Anette Fält, Charlotte Fält, Karin Enell Smith. Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1593.

Type I interferon/IRF7 axis instigates chemotherapy-induced immunological dormancy in breast cancer

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER−) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER− breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-β/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4+/CD8+ T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-β production. Presence of IFN-β in the circulation of ER− breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER− breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-β/IFNAR pathway in this effect. Further, IFN-β emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER− breast cancer patients treated with (neo)adjuvant chemotherapy.

Abstract 5746: CRISPR9-mediated FGL2-KO in tumor cells impairs tumor progression in brain and triggers antitumor immune memory systematically via NF-κB dependent Batf3 expression in DCs

Abstract Brain cancer is the 10th leading cause of death in United States with increasing primary tumor morbidity and secondary brain metastasis morbidity. Glioblastoma (GBM) is most lethal brain tumor with less than 5% 5 years survival rate marked by significant resistance to majority of therapies targeting tumor cells or tumor microenvironment. Here we shown one approach to convert aggressive GBM cells to immunogenic tumor cells by knockout FGL2 in GBM cells to evoke efficient anti-tumor immunosurvilliance. FGL2 is an immunosuppressive molecule and its expression level is strongly correlated with glioma grade and poor diagnosis. FGL2 from GBM cells, but not from host cells, is essential for immune evasion dependent GBM progression using both preclinical GBM and brain metastatic tumor models. Knockout FGL2 in GBM cells completely abolish tumor progression of GBM models challenged with low or high number of tumor cells in immunocompetent mice and protect mice from tumor rechallenge in periphery. This eradication effect is impaired in immune deficient mice and CD8+ T cells depleted mice, but not in FGL2KO mice, showing the necessarily of immune system to eliminate FGL2KO tumor cells and the requirement of FGL2 in tumor cells to evade immunosurvilliance. To understand how the systemic CD8+ T cell-dependent anti-tumor response and memory was induced by FGL2KO GBM cells, we studied the impact of FGL2KO tumor cells on dendritic cells (DCs) which control immune response via its critical role in the generation of effective cytotoxic T lymphocyte (CTL) responses. Knockout FGL2 in GBM cells induced CD8a+/CD103+ DCs accumulation in brain in vivo; DCs proliferation and CD103+ DCs induction in vitro. In agreement with this discovery, knockout Batf3, a key transcription factor regulating CD8a+/CD103+ DCs, reverse the impact of FGL2KO in GBM cell in vivo. By molecule analysis, a novel signaling pathway was revealed that knockout FGL2 in GBM cells induce NF-κ B activation, and this activation induced Batf3 and associated CD103 expression in DCs. Therefore, target specific molecule in GBM cells, such as knockout FGL2, can convert aggressive GBM cells to immunogenic cells, pointing to new approach for immune therapy. Citation Format: Jun Yan, Qingnan Zhao, Konrad Gabrusiewicz, Ling-Yuan Kong, Longfei Huo, Xueqing Xia, Jun Wei, Jingda Xu, Eric Davis, Amy B. Heimberger, Shulin Li. CRISPR9-mediated FGL2-KO in tumor cells impairs tumor progression in brain and triggers antitumor immune memory systematically via NF-κB dependent Batf3 expression in DCs [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 5746.

B cells promote tumor progression in a mouse model of HPV-mediated cervical cancer.

Enhancing anti-tumor immunity and preventing tumor escape are efficient strategies to increase the efficacy of therapeutic cancer vaccines. However, the treatment of advanced tumors remains difficult, mainly due to the immunosuppressive tumor microenvironment. Regulatory T cells and myeloid-derived suppressor cells have been extensively studied, and their role in suppressing tumor immunity is now well established. In contrast, the role of B lymphocytes in tumor immunity remains unclear because B cells can promote tumor immunity or display regulatory functions to control excessive inflammation, mainly through IL-10 secretion. Here, in a mouse model of HPV-related cancer, we demonstrate that B cells accumulated in the draining lymph node of tumor-bearing mice, due to a prolonged survival, and showed a decreased expression of MHC class II and CD86 molecules and an increased expression of Ly6A/E, PD-L1 and CD39, suggesting potential immunoregulatory properties. However, B cells from tumor-bearing mice did not show an increased ability to secrete IL-10 and a deficiency in IL-10 production did not impair tumor growth. In contrast, in B cell-deficient μMT mice, tumor rejection occurred due to a strong T cell-dependent anti-tumor response. Genetic analysis based on single nucleotide polymorphisms identified genetic variants associated with tumor rejection in μMT mice, which could potentially affect reactive oxygen species production and NK cell activity. Our results demonstrate that B cells play a detrimental role in anti-tumor immunity and suggest that targeting B cells could enhance the anti-tumor response and improve the efficacy of therapeutic cancer vaccines.

Abstract A103: Intracellular immunotherapy of disseminated pancreatic cancer activates potent IFNγ and CD8+ T cell dependent anti-tumor responses

Abstract Innovative therapies with the ability to activate effective cellular-based immune mechanisms in conjunction with current treatment strategies have potential to combat highly aggressive solid and metastatic tumors such as pancreatic adenocarcinoma, which remains one of the most deadly cancers with an overall 5-year survival rate of 6% following diagnosis. In this study, we explored the immunotherapeutic mechanisms triggered by an avirulent and nonreplicating obligate intracellular parasite Toxoplasma gondii (cps strain), which preferentially invades myeloid cell types, in the disseminated pancreatic tumor environment. We hypothesized the strong induction of Th1 responses in the tumor environment triggered by cps may stimulate significant mechanisms that could halt or eliminate established pancreatic cancer. Pan02 cells were injected intraperitoneally into syngeneic C57BL/6 mice. Seven days later, these mice were treated with 2, 3, or 5 doses of cps or PBS, as a treatment control. Pan02 tumor-bearing mice treated with cps had a significant survival advantage over untreated mice. Additionally, treatment with heat-killed cps, which is unable to actively invade, led to loss of treatment efficacy. Following a single cps treatment CD11b+CD11c+ dendritic cell populations increased and CD11b+F4/80+CD11c- macrophage populations decreased in the peritoneum. Cps-invaded macrophages and dendritic cells in the tumor environment upregulated surface expression of CD80 and CD86, and there was an increase in local production of IL-12 up to 7 days post-treatment. Pan02 tumor-bearing mice unable to produce IL-12 lost protection mediated by cps treatment. Seven days post-cps treatment, both CD4+ and CD8+ T cells were significantly higher than within the tumor microenvironment of untreated mice. Local T cells had lower expression of CD62L and increased expression of CD44 and CD69 indicating activation of recruited T cells. Six and seven days post-cps treatment, local IFNγ production in Pan02 tumor-bearing mice increased and the numbers of IFNγ+ CD4+ and IFNγ+ CD8+ T cells were higher. Similar to IL-12, loss of IFNγ within the tumor microenvironment led to a loss of treatment efficacy. A lack of CD8+ T cells prior to and during therapy resulted in a loss of treatment efficacy, whereas CD4+ T cells and NK cells were not required for treatment efficacy. CD8+ T cells isolated from the spleens of treated animals 10 days after cps treatment robustly produced IFNγ ex-vivo when stimulated with Pan02 cells. This response appears to be tumor specific as stimulation with irrelevant tumor antigen did not induce an IFNγ response from the same CD8+ T cells. Cps-mediated activation of tumor-associated myeloid cells leads to increases in local IFNγ+ CD8+ T cells, which drive specific anti-tumor responses. Cps induction of host Th1 responses elicited in the tumor microenvironment demonstrates the capability of cps to be broadly applied as an immunotherapeutic against pancreatic tumors. Citation Format: Kiah L. Sanders, Barbara A. Fox, David J. Bzik. Intracellular immunotherapy of disseminated pancreatic cancer activates potent IFNγ and CD8+ T cell dependent anti-tumor responses. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A103.

Interleukin-18 synergism with interleukin-2 in cytotoxicity and NKG2D expression of human natural killer cells.

Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is an immunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administrated with other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity as well as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for 48 h with IL-18 and IL-2, then CD107a expression on CD3-CD56+ NK cells was determined by three-colour flow cytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human colon carcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells. The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with using IL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement of NK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition, blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combining IL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance the cytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for the use of combined IL-18 and IL-2 in tumor immunotherapy.

Abstract A86: Inactivation of p110delta PI3K releases potent antitumor immunity.

Abstract In cancer the tumor suppressive potential of the immune system is limited by the induction of self-tolerance and chronic inflammation promoted through the activity of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs) respectively. The phosphoinositide-3-kinase (PI3K) pathway is very frequently activated in cancer cells, and clinical trials with PI3K inhibitors are ongoing. The p110delta isoform of PI3K is highly enriched in leukocytes in which it regulates various immune functions. p110delta is involved in both the adaptive and innate immune system and p110delta inhibitors may be used to treat a broad range of immune disorders including T cell mediated auto-immune disorders. An expected side effect of long-term p110delta inhibition is, immune suppression, which could pre-dispose to cancer. We now show that paradoxically, genetic or pharmacologic inactivation of p110delta protects mice against tumor growth and metastasis. Blockade of p110delta; inhibits the activation of Treg cells and the accumulation of MDSCs, thus allowing the development of an effective CD8+ T cell-dependent anti-tumor response. These data provide a rationale for the use of p110delta inhibitors as adjuvants to boost anti-tumor immune responses in cancer. Note: This abstract was not presented at the conference. Citation Format: Khaled Ali, Dalya Soond, Roberto Pinero, Wayne Pearce, Cheryl Scudamore, Lori Friedman, Martin Turner, Klaus Okkenhaug, Bart Vanhaesebroeck. Inactivation of p110delta PI3K releases potent antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A86.

Knockdown of HMGB1 in Tumor Cells Attenuates Their Ability To Induce Regulatory T Cells and Uncovers Naturally Acquired CD8 T Cell-Dependent Antitumor Immunity

Although high mobility group box 1 (HMGB1) in tumor cells is involved in many aspects of tumor progression, its role in tumor immune suppression remains elusive. Host cell-derived IL-10 suppressed a naturally acquired CD8 T cell-dependent antitumor response. The suppressive activity of tumor-associated Foxp3(+)CD4(+)CD25(+) regulatory T cells (Treg) was IL-10 dependent. Neutralizing HMGB1 impaired tumor cell-promoted IL-10 production by Treg. Short hairpin RNA-mediated knockdown of HMGB1 (HMGB1 KD) in tumor cells did not affect tumor cell growth but uncovered naturally acquired long-lasting tumor-specific IFN-γ- or TNF-α-producing CD8 T cell responses and attenuated their ability to induce Treg, leading to naturally acquired CD8 T cell- or IFN-γ-dependent tumor rejection. The data suggest that tumor cell-derived HMGB1 may suppress naturally acquired CD8 T cell-dependent antitumor immunity via enhancing Treg to produce IL-10, which is necessary for Treg-mediated immune suppression.