Plate in petri dish + M-CSF Isolate CD14+CD16- Monocytes from blood Add more media + M-CSF Day-3 Day-7 Remove supernatant & wash adherent cells with PBS BMDM (M0) IFNg + LPS M1 Macrophages Isolate CD14+CD16- Monocytes from blood Plate cells + GMCSF + IL4 Day-3 Spin cells Change media GMCSF + IL4 +LPS Day-5 Day-7 Immature DCs Mature DCs ATYR1923 Reduces Neutrophil Infiltration in an Acute Lipopolysaccharide (LPS) Lung Injury Model Suzanne Paz, Clara Polizzi, Dalena Chu, Lauren Guy, Christoph Burkart, Ryan Adams, David King, Kathleen Ogilvie, Sanna Rosengren aTyr Pharma, San Diego, CA, USA Neutrophils (CD45+CD11b+Siglec-F-CD11c-GR1+CX3CR1-Ly6G+) FSC Live/Dead FSC CD45 FSC CD11b Live Cells Immune Cells Myeloid Cells CD11c Siglec-F CD11c Siglec-F F4/80 CD64 Alveolar Macrophages (AM) CX3CR1 Gr1 Ly6G GR1 Monocytes (CD45+CD11b+Siglec-F-CD11c-GR1+CX3CR1+) AM (CD45+CD11b+Singlec-F+CD11c+CD64+F4/80+) AM Other Myeloid Cells Abstract Introduction In vivo Experimental Procedure A number of aminoacyl tRNA-synthetases have evolved non-canonical functions including the tRNA synthetase for histidine, HARS. HARS downregulates immune responses via its N-terminal domain, which we have termed the iMod (immunomodulatory) domain. The iMod domain was fused to human IgG1 Fc to generate ATYR1923, which is currently in clinical evaluation for pulmonary sarcoidosis. ATYR1923 binds to neuropilin-2 (NRP-2), a pleiotropic co-receptor participating in several pathways including class III semaphorins/plexins and VEGF-C/VEGFR3. To date, little is known about the role of NRP-2 in immune regulation, although growing evidence indicates that NRP-2 influences myeloid cell biology such as activation and recruitment to inflammatory sites. For instance, NRP-2 expression on alveolar macrophages regulates airway inflammatory responses to inhaled LPS (Immormino et al. 2018). To determine whether ATYR1923 was able to influence myeloid cell migration, ATYR1923 or a pentameric iMod construct, iMod-COMP, were administered intravenously to C57BL/6 mice 24h prior to LPS challenge, either by intraperitoneal (IP) injection to induce a systemic inflammatory response or by airway administration to generate acute lung inflammation. Multi-color flow cytometry was used for immunophenotyping analysis and detection of NRP-2 levels on surfaces of various immune cell populations. In vitro, mouse bone-marrow derived macrophages (BMDM), human THP-1 monocytic cells, and primary human dendritic cells (DCs) were used to confirm NRP-2 cell surface expression. Results indicated that LPS stimulation in vitro or in vivo upregulated NRP-2 on a variety of myeloid cells including macrophages (splenic & alveolar), DCs and neutrophils. Notably, prophylactic administration of ATYR1923 or iMod.COMP led to a significant and dose-dependent reduction in LPS-induced neutrophil infiltration into the bronchoalveolar space. This finding appeared to be specific to neutrophil trafficking, since the number of monocytes, alveolar macrophages, or other myeloid cells was not altered. Altogether, these results suggest that the activity of ATYR1923 includes inhibition of neutrophil migration to inhibit lung inflammation. Group Test Article (TA) Dose (mg/kg) TA Route LPS Treatment (µg) Time Point (hrs) 1 NA       24 2 Vehicle 0 IV 0 24 3 Vehicle 0 IV 10 24 4 ATYR1923 1 IV 10 24 5 ATYR1923 3 IV 10 24 6 ATYR1923 10 IV 10 24 7 iMod-comp 3 IV 10 24 A number of non-canonical functions of proteins generated from tRNA synthetase genes have been reported, demonstrating diverse roles for these proteins outside of protein synthesis (Wakasugi & Schimmel, 1999; Park et al., 2008; Arif et al., 2017). Proteins derived from the histidyl-tRNA synthetase (HARS) gene are found extracellularly and are thought to play a role in regulating key cells in the immune system to ensure appropriate control of immune responses. ATYR1923 is a clinical stage immunomodulatory protein. ATYR1923 comprises the iMod domain of HARS fused to Human IgG Fc (Fig 1.) to extend plasma half-life. NRP-2 was identified to be a binding partner of ATY1923. ATYR1923 exerts some of its immunomodulatory functions by affecting T cell activation & cytokine release (data presented at AAI 2018 by E. Mertcshing). NRP-2 was shown to play a role in airway inflammatory responses to inhaled LPS (Immormino et al. 2018) Day -1: Body weights (BW) for TA administration were recorded & animals were dosed with TA at 5ml/kg according to the table above Day 0: Mice in grps 3-7 were anesthetized at 2-4% Isoflurane (1L/min.) & dosed OP (oropharyngeally) with 10 ug LPS in 50 µL PBS. Grp 2 received 50 µL PBS only. Grp 1 mice were naïve to induction & treatment. Day 1: BWs were recorded and mice were euthanized with lethal ketamine/xylazine cocktail (~300/30 mg/kg) at 24 hours post LPS induction. Blood was collected from the abdominal vein, processed for serum for pathway analysis (cytokine, ATYR1923 &/or NRP2 levels). The lung and trachea were exposed & perfused for BALF (bronchoalveolar lavage) collection using 0.8 ml PBS through cannulated trachea. Collected BALF was placed on ice and volume recorded. BALF cells were collected by centrifugation and supernatant was retained for potential measurement of pathway analysis (HARS pathway proteins, cytokines and/or ATYR1923 etc.). RBC lysis was applied to BALF cells & stained for flow cytometry analysis. Results from flow analysis were analyzed using FlowJo & statistical analysis performed using Prism. Results In vitro Experimental Procedure Fig. 3 Generation of mouse BMDM Fig 4. Generation of Human Macrophages from THP1 Cell Line Fig 5. Generation of Human Primary Macrophages Fig 6. Generation of Human Primary Dendritic Cells Fig 2. Flow Cytometry Gating Strategy Flush cells in IMDM Spin Cells Plate in petri dish + M-CSF Collect BM Add more RPMI + M-CSF Day-3 Remove supernatant & wash adherent cells with PBS Day-7 BMDM (M0) IFNg + LPS M1 Macrophages THP-1 cells (suspension) Monocytes/M0 M1 Macrophages (adherent) (24 hours) M0 Macrophages (adherent) IFNg + LPS +PMA OR LPS + IFNg +PMA (24 hours) +PMA Fig. 7 ATYR1923 Binds Human and Mouse NRP-2 Conclusions Anticodon-Binding Domain iMod Domain Aminoacylation Domain Histidyl-tRNA Synthetase HARS (1-509) ATYR1923 iMod (2-60)-Fc iMod (2-60) Human IgG1 Fc iMod Domain iMod-COMP ATYR1923 binds to both human and mouse NRP-2. NRP-2 is detected on the cell surface of myeloid cells both in vitro and in vivo. NRP-2 was induced following activation of TLR found on the cell surface (mainly TLR1, 2, 4, 5 & 6), but not endosomal TLR ligands (TLR3, 7/8, & 9). In an acute LPS lung injury model, a significant increase in immune cell infiltration and NRP-2 expression was observed in positive control group. ATYR1923 significantly decreased the CD11b+ population following LPS installation in the lung, which was ascribed to an inhibitory effect on neutrophil infiltration. These findings highlight the potential of ATYR1923 to regulate myeloid cell biology during lung inflammation. Fig 1. Generation of HARS-Derived Proteins Fig. 1 Schematic representation of HARS, iMod domain, ATYR1923 & iMod-COMP Fig. 2 Flow cytometry dot plots demonstrating the gating strategy utilized to identify immune cells, myeloid cells, alveolar macrophages (AM), monocytes and neutrophils. Table 1. In vivo study design Fig.3-6 Schematic representations showing the experimental procedures utilized to generate: mouse Bone Marrow Derived Macrophages (Fig 3); human macrophages from THP1 cell line (Fig 4); human primary macrophages from monocytes (Fig 5); human primary dendritic cells (Fig 6). Fig. 9 ATYR1923 Reduces Neutrophil Infiltration in BALF Fig 9. Results obtained from BALF Immunophenotyping A) Dot plot and immune cell count. B) Dot plot, Myeloid cell count and NRP-2 .C) Dot, plot, Monocyte cell count and NRP-2 expression. D) Dot plot, Neutrophil count and NRP-2 expression. E) Soluble NRP2 in BALF. F) Levels of ATYR1923 detected in BALF. G) Neutrophil counts across three independent studies H) NRP-2 expression across various Myeloid cells. Mean values +/- SEM. Statistical analysis: one-Way ANOVA; Dunnett’s Multiple Comparison. Control LPS FSL-1 HKLM Pam3CSK4 ST-FLA Control LPS Poly(I:C) LMW ssRNA40 Poly(I:C) HMW CpG B Imiquimod Control Mouse BMDM THP-1 Cell Line M1 M0 Control M1 M0 Human Primary Macrophages M1 M0 Control Day 7 Day 3 Control Human Primary Dendritic Cells Fig. 8 In Vitro NRP-2 Expression A) B) C) D) E) Fig 8. Histogram representation of NRP-2 expression detected on the cell surface of various in vitro generated myeloid cells. A) M0 and M1 macrophages generated from mouse bone marrow cells. B) M0 and M1 macrophages generated from human THP-1 cell line. C) M0 and M1 macrophages generated from primary human monocytes. D) Day 3 and Day7 of human dendritic cells differentiated from primary monocytes. E) M0 Human THP-1 cells stimulated with various TLR ligands as indicated. THP-1 Cell Line References: Arif A, Terenzi F, Potdar AA, Jia J, Sacks J, China A, Halawani D, Vasu K, Li X, Brown JM, Chen J, Kozma SC, Thomas G & Fox PL (2017) EPRS is a critical mTORC1-S6K1 effector that influences adiposity in mice. Nature 542, 357-361. Park SG, Schimmel P & Kim S (2008) Aminoacyl tRNA synthetases and their connections to disease. Proc Natl. Acad. Sci. 105, 11043-11048. Wakasugi K & Schimmel P (1999) Two distinct cytokines released from a human aminoacyl-tRNA synthetase. Science 284, 147-151. Immormino RM, Lauzier DC, Nakano H, Hernandez ML, Alexis NE, Ghio AJ, Tilley SL, Doerschuk CM, Peden DB, Cook DN & Moran TP. (2018) Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide. Am J Physiol Lung Cell Mol Physiol. 315 (2): L202-L211.111v a1 a2 b1 b2 c NRP2 A) B) C) D) E) F) G) H) NRP-2 is a pleiotropic receptor with important functional roles in cardiovascular, nervous and immune systems (Guo HF et al., 2015) NRP-2 is a type I transmembrane protein that acts as a co-receptor for class III semaphorins (SEMA3) and vascular endothelial growth factor C (VEGF-C) and Plexins (Roy et al. 2017). NRP-2 can be found on the cell surface or in soluble form created by either alternative splicing or shedding (Rosignol et al. 2000; Roy et al. 2017) Acknowledgements: The authors would like to thank Jeanette Ampudia, Elisabeth Mertsching, Luke Burman and Kristina Hamel for their contributions to this poster. Fig 7. A) Schematic representation of NRP-2 showing various domains (a1, a2, b1, b2 and c). B) EC50 curves of ATYR1923 binding to human NRP-2 or mouse NRP-2 in a HEK293 overexpressing system; N15 is an Fc control for ATYR1923. %ATYR1923+ cells was identified by flow cytometry based binding assay. A) B) Guo HF, Vander Kooi CW. (2015) Neuropilin Functions as an Essential Cell Surface Receptor. JBC 290: 29120-29126. Rossignol M, Gagnon ML, Klagsbrun M. (2000) Genomic organization of human neuropilin-1 and neuropilin-2 genes: identification and distribution of splice variants and soluble forms. Genomics 70: 211-222. Roy S, Bag AK, Singh RK, Talmadge JE, Batra SK, Datta K. (2017) Multifaceted Role of Neuropilins in the Immune System: Potential Targets for Immunotherapy. Front. Immunol. 8:1228. Exhibit 99.2