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In the current study, we assessed the effects of conventional and Ultra High Dose Rate (UHDR) irradiation on behavioral and cognitive performance one month following exposure and assessed whether these effects were associated with alterations in the number of immune cells in the hippocampus using flow cytometry. Conventional- and UHDR-irradiated mice showed impaired novel object recognition. During fear learning, conventional- and UHDR-irradiated mice moved less during the inter-stimulus-interval (ISI) and UHDR-irradiated mice also moved less during the baseline period (prior to the first tone). In irradiated mice, reduced activity levels were also seen in the home cage; conventional- and UHDR-irradiated mice moved less during the light period and UHDR-irradiated mice moved less during the dark period. Following behavioral and cognitive testing, infiltrating immune cells in the hippocampus were analyzed by flow cytometry. The percent Ly6G+ CD45+ cells in the hippocampus was lower in conventional- and UHDR-irradiated than sham-irradiated mice, suggesting that neutrophils might be particularly sensitive to radiation. The percent of Ly6G+ CD45+ cells in the hippocampus was positively correlated with the time spent exploring the novel object in the object recognition test, suggesting that the reduced percent Ly6G+ CD45+ cells in the hippocampus might mediate some of the detrimental radiation-induced cognitive effects.

BackgroundThe tumor microenvironment (TME) often contains high levels of suppressive myeloid cells that contribute to innate checkpoint inhibitor (CPI) resistance. Pionyr’s Myeloid Tuning approach involves altering the composition and/or the function of myeloid cells in the TME. Myeloid reprogramming alters the function of immunosuppressive myeloid cells to acquire an immunostimulatory phenotype. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor enriched on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, Pionyr developed an afucosylated humanized anti-TREM1 monoclonal antibody termed PY159 and characterized it in pre-clinical and translational biomarker assays described below.MethodsPY159 responses in human whole blood and dissociated primary tumor cells in vitro were evaluated by flow cytometry and measurement of secreted cytokines and chemokines by MSD. TREM1 expression in human tumors was validated by scRNAseq, flow cytometry, and immunohistochemistry (IHC). In vivo efficacy and pharmacodynamic studies of a surrogate anti-mouse TREM1 antibody, termed PY159m, were evaluated using syngeneic mouse tumor models, either as a single-agent or in combination with anti-PD-1. To select tumor types and patients most likely to benefit from PY159 therapy, Pionyr developed qualitative and quantitative monoplex and multiplex IHC assays that detect TREM1 expression levels in human tumor tissues.ResultsPY159 treatment in vitro induced signaling, upregulated monocyte activation markers, and induced proinflammatory cytokines. In human tumors, TREM1 was detected on tumor-associated neutrophils, tumor-associated macrophages, and monocytic myeloid-derived suppressive cells. The surrogate PY159m anti-mouse TREM1 antibody exhibited anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with anti-PD-1. Screening for TREM1 expression in tumor tissues demonstrated that TREM1+ tumor associated myeloid cells were highly enriched in the TME of multiple solid tumor indications. The monoplex and multiplex IHC assays offered insights into the localization of TREM1+ myeloid cells and their spatial relationship with other immune cells present in the TME to determine what immune composition will be more favorable for response to PY159 therapy.ConclusionsCollectively, the available nonclinical data support PY159 as a TREM1 agonist that reprograms myeloid cells and unleashes anti-tumor immunity. PY159 safety and efficacy are currently being evaluated in first-in-human clinical trial (NCT04682431) involving select advanced solid tumors patients resistant and refractory to standard of care therapies alone and in combination with a CPI. The TREM1 IHC assay is successfully being used on FFPE archival tumor tissues from enrolled patients to determine TREM1 expression levels.

Background Tumor-associated myeloid cells can impede productive anti-tumor immunity. One strategy for targeting immunosuppression is myeloid reprogramming, which drives immunosuppressive myeloid cells to acquire an immunostimulatory phenotype. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor expressed on neutrophils and subsets of monocytes and tissue macrophages. TREM1 associates with DAP12 adaptor and induces proinflammatory signaling, amplifies innate immune responses, and is implicated in the development of acute and chronic inflammatory diseases. TREM1 is also enriched in tumors, specifically on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, we developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody, and characterized it in the pre-clinical assays described below. Materials and Methods An FcγR binding ELISA and a Jurkat TREM1/DAP12 NFAT-luciferase reporter cell line were used to assess PY159 binding to human FcγRs and TREM1 signaling, respectively. PY159 responses in human whole blood in vitro were evaluated by flow cytometry, transcriptional analysis of sorted leukocyte subsets, and measurement of secreted cytokines/chemokines by MSD. A Transwell system was used to evaluate PY159 effects on neutrophil chemotaxis. TREM1 expression in human tumors was validated by scRNAseq, immunohistochemistry, and flow cytometry. Anti-tumor efficacy of a surrogate anti-mouse TREM1 antibody, PY159m, was evaluated using syngeneic mouse tumor models, either as a single agent or in combination with anti-PD-1. Results PY159 afucosylation increased its binding affinity for FcγR and its ability to activate TREM1/DAP12 signaling. In human blood assays, PY159 treatment did not induce depletion of TREM1-expressing cells. Rather, it upregulated monocyte activation markers, promoted neutrophil chemotaxis, and induced proinflammatory cytokines and chemokines, which was dependent on PY159 afucosylation. In human tumors, TREM1 was detected on tumor-associated neutrophils, tumor-associated macrophages, and monocytic myeloid-derived suppressive cells. PY159 induced proinflammatory cytokines and chemokines in dissociated human tumors in vitro, demonstrating that PY159 can reprogram tumor-associated myeloid cells. A surrogate anti-mouse TREM1 antibody, PY159m, exhibited anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with anti-PD-1. Conclusions These results show that PY159 is a TREM1 agonist that reprograms myeloid cells and unleashes anti-tumor immunity. PY159 safety and efficacy are currently being evaluated in first-in-human clinical trial (NCT04682431) involving patients resistant and refractory to standard of care therapies. Disclosure Information V. Juric: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. E. Mayes: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. M. Binnewies: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. P. Canaday: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. T. Lee: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. S. Dash: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. J.L. Pollack: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. J. Rudolph: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. V. Huang: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. X. Du: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. N. Jahchan: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. A.J. Ramoth: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. S. Mankikar: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. M. Norng: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. C. Santamaria: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. K.P. Baker: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc. L. Liang: A. Employment (full or part-time); Significant; Pionyr Immunotherapeutics Inc.

Summary: Converting checkpoint inhibitor (CPI)-resistant individuals to being responsive requires identifying suppressive mechanisms. We identify TREM2+ tumor-associated macrophages (TAMs) as being correlated with exhausted CD8+ tumor-infiltrating lymphocytes (TILs) in mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc domain-enhanced anti-TREM2 monoclonal antibody (mAb) therapy promotes anti-tumor immunity by elimination and modulation of TAM populations, which leads to enhanced CD8+ TIL infiltration and effector function. TREM2+ TAMs are most enriched in individuals with ovarian cancer, where TREM2 expression corresponds to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drives potent anti-tumor immunity. These results highlight TREM2 as a highly attractive target for immunotherapy modulation in individuals who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

Converting checkpoint inhibitor (CPI) resistant patients to responsive requires identifying novel suppressive mechanisms. We identified TREM2 + tumor associated macrophages (TAMs) as being correlated with exhausted CD8 + tumor infiltrating lymphocytes (TILs) in both mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc-domain enhanced anti-TREM2 monoclonal antibody (mAb) therapy promoted anti-tumor immunity by elimination and modulation of TAM populations which led to enhanced CD8 + TIL infiltration and effector function. TREM2 + TAMs were most enriched in human ovarian cancer patients, where TREM2 expression corresponded to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drove potent anti-tumor immunity. Together, these results highlight TREM2 as a highly attractive target for immunotherapeutic modulation in patients who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

Myeloid cells present in the tumor microenvironment can exist in immunosuppressive states that impede productive anti-tumor immunity. One strategy for targeting these immunosuppressive mechanisms is reprogramming of myeloid cells from immunosuppressive to immunostimulatory, resulting in the removal of the immune inhibition and unleashing of anti-tumor immunity. Triggering receptor expressed on myeloid cells-1 (TREM1) is an immunoglobulin superfamily cell surface receptor expressed primarily on neutrophils and subsets of monocytes and tissue macrophages. TREM1 signals through the association with DAP12 adaptor protein and mediates proinflammatory signaling, amplifies the host immune response to microbial pathogens, and has been implicated in the development of acute and chronic inflammatory diseases. TREM1 is also enriched in tumors, specifically on tumor-associated myeloid cells. To investigate the potential of TREM1 modulation as an anti-cancer therapeutic strategy, we developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody. We found that PY159 does not deplete TREM1-expressing cells, but rather acts as a TREM1 agonist. In vitro human blood assays showed that PY159 treatment upregulated activation markers on monocytes and stimulated neutrophil chemotaxis, as assayed by flow cytometry, transcriptional analysis, and in vitro migration assays. Furthermore, PY159 induced a selective set of proinflammatory cytokines and chemokines, which was dependent on PY159 afucosylation. We validated TREM1 expression in human tumors by single-cell RNAseq, immunohistochemistry, and flow cytometry, and found that it is expressed on myeloid populations, including tumor-associated neutrophils (TAN), tumor-associated macrophages (TAM), and monocytic myeloid-derived suppressive cells (mMDSC). We showed that PY159 can also induce proinflammatory cytokines and chemokines in dissociated human tumors in vitro, demonstrating that PY159 can reprogram tumor-associated myeloid cells. Finally, in vivo treatment of mice with a surrogate anti-mouse TREM1 antibody, PY159m, promoted anti-tumor efficacy in several syngeneic mouse tumor models, both as single-agent and in combination with checkpoint inhibitors, such as anti-PD-1 antibody. Together, these results demonstrate that therapeutic targeting of TREM1 with a TREM1 agonist antibody, PY159, promotes myeloid cell reprogramming and anti-tumor immunity. PY159 safety and tolerability have been demonstrated in non-human primates, and safety and efficacy of PY159 are currently being evaluated in first-in-human clinical trial (NCT04682431) including solid tumors that are resistant and refractory to standard of care therapies. Citation Format: Erin Mayes, Vladi Juric, Mikhail Binnewies, Pamela Canaday, Tian Lee, Subhadra Dash, Joshua L. Pollack, Joshua Rudolph, Vicky Huang, Xiaoyan Du, Nadine Jahchan, Asa J. Ramoth, Shilpa Mankikar, Manith Norng, Carlos Santamaria, Kevin P. Baker, Linda Liang. Therapeutic targeting of TREM1 with PY159 promotes myeloid cell reprogramming and unleashes anti-tumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P104.