Slit guidance ligand 2 (SLIT2) is a large, secreted protein which binds roundabout (ROBO) receptors on multiple cell types including neurons and kidney podocytes. SLIT2-ROBO2 mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP-Gremlin signaling also regulates UB formation. However, direct crosstalk between the ROBO2-SLIT2 and BMP-Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP-Gremlin signaling pathways. We found that the SLIT2-Gremlin interaction inhibited both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 downregulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMP-mediated repression of SLIT2.

Background:Activation and expansion of regulatory T cells (Tregs) has been proposed as a strategy to treat autoimmunity. When administered in low doses, IL-2 expands and activates Tregs leading to clinical response in several autoimmune diseases. However, the narrow therapeutic window of IL-2 results in loss of selectivity for Tregs and concurrent activation of conventional T cells (Tconv) and NK cells, limiting its clinical utility. This loss of selectivity may negate the clinical benefit of Treg activation and lead to dose-limiting side effects. PT101 is a novel engineered variant of IL-2 fused to an Fc protein backbone which in preclinical studies selectively activates Tregs without expanding Tconv or NK cells. PT101 is in clinical development for the treatment of patients with autoimmune diseases.Objectives:To assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of PT101 after a single dose in healthy human volunteers.Methods:We conducted a randomized, double-blind, single-ascending dose trial of PT101 or placebo (3:1 allocation). Five dose levels from 1 mg to 10 mg were administered by subcutaneous injection. Adverse events, physical examination findings, and clinical laboratory results were assessed for 29 days. Serum PT101 levels and antidrug antibody were assessed. Changes in mononuclear cell populations were measured in peripheral blood by flow cytometry.Results:56 subjects were administered PT101 or placebo. All subjects completed the study. There were no deaths, serious adverse events, dose limiting toxicities, or clinically significant changes in vital sign, ECG, or laboratory results. All adverse events were Grade 1 or 2 and self-limited. Injection site reactions were the most common adverse event. Transient increases in eosinophil counts were observed in some subjects, consistent with the known class effect of IL-2. Peak levels of PT101 occurred 11.0 to 14.6 hours after administration, and declined with a mean half-life of 20.4 to 28.3 hours, demonstrating linear exposure through the dose range. No anti-drug antibodies were induced. PT101 caused dose-related expansion of Tregs that plateaued at doses between 3.5 and 10 mg. Mean maximum expansion above baseline was 3.6-fold for total Tregs and 72.5-fold for the CD25bright subset of Tregs. Maximal expansion was observed by Day 8-10 with a return toward baseline by Day 29. Over 80% of subjects achieved a 2-fold or greater expansion of total Tregs (Table 1). No significant expansion of Tconv or NK cells was observed at any dose level.Table 1.Percent Total Treg RespondersFold Change Total TregsPlacebo(n=14)1 mg(n=6)3.5 mg(n=12)5 mg(n=12)7.5 mg(n=6)10 mg(n=6)≥ 2X7%33%83%83%100%100%≥ 3X0%0%58%75%33%50%≥ 4X0%0%24%42%33%17%Conclusion:PT101 was safe and well tolerated after a single dose in healthy volunteers. Marked expansion of both total Treg and CD25bright Treg cells was observed. High selectivity for Tregs was observed with no significant expansion of pro-inflammatory Tconv and NK cells even at the highest dose studied. These results support the therapeutic potential of PT101 in planned multiple dose studies in systemic lupus erythematosus, ulcerative colitis, and other autoimmune diseases.References:[1]Klatzmann, D., Abbas, A. The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases. Nat Rev Immunol 15, 283–294 (2015)Acknowledgements:Pandion Therapeutics acknowledges the participants and research staff who contributed to this clinical trialDisclosure of Interests:John S Sundy Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Kevin L. Otipoby Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Nathan Higginson-Scott Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Jyothsna Visweswaraiah Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Erik Sampson Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Katalin Kis-Toth Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Adrianne Monsef Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Parika Petaipimol Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, David Essayan Consultant of: Pandion Therapeutics, Mary Ellen Cosenza Consultant of: Pandion Therapeutics, Rahul Kakkar Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics, Jo Viney Shareholder of: Pandion Therapeutics, Employee of: Pandion Therapeutics

Background:Regulatory T cells (Tregs) play a critical role in immune homeostasis and are dysfunctional in many autoimmune diseases. Interleukin 2 (IL-2) drives the proliferation and function of Tregs. via the heterotrimeric IL-2 receptor (CD25/CD122/CD132). As a result, CD25 loss-of-function in mice is associated with Treg deficiency and widespread autoimmunity. Low dose IL-2 is being evaluated for treatment of autoimmune diseases and has been shown to expand Tregs, however it has a narrow selectivity window before activating conventional T cells and natural killer cells. To enhance IL-2 selectivity and improve its therapeutic utility for activating and expanding Tregs, mutations can be introduced that reduce CD122/CD132 affinity thus creating a dependency on CD25 binding for signaling through CD122/CD132 upon IL-2 facilitated CD25/CD122/CD132 trimer formation.Objectives:To generate a highly selective IL-2 mutein that activates and expands Tregs selectively that can be used for treatment of autoimmune diseasesMethods:Using a structure guided approach, we introduced mutations in IL-2 that significantly decreased CD122 binding affinity in addition to other mutations that increased CD25 binding affinity. Finally, we explored additional mutations, format, orientation, and linker lengths to generate the most potent, selective molecule with drug-like manufacturability. These structure activity relationship efforts culminated in the generation of PT101, a mutant IL-2 Fc fusion that is selective in activating and expanding Tregs in vitro and in vivo.Results:PT101 selectively induced STAT5 phosphorylation in human and cynomolgus monkey Tregs in vitro. In humanized NOD-scid IL-2Rg-null (NSG) mice and cynomolgus monkeys, administration of PT101 dose-dependently and selectively expanded Tregs without significant effects on other immune cell types, and without eliciting proinflammatory cytokine production. The Tregs from PT101-dosed humanized mice have increased expression of FOXP3 and CD25, suggesting enhanced function and stability. In a Phase 1a single ascending dose clinical trial, PT101 was well-tolerated and selectively expanded total Tregs, with a mean maximal increase of up to 3.6-fold over baseline in healthy volunteers. There was no evidence of expansion of natural killer cells nor pro-inflammatory conventional T cells at any of the doses studied.Conclusion:PT101 selectively activated and expanded Tregs without significant effects on other immune cell types, and without eliciting proinflammatory cytokine production. These Tregs have enhanced function and stability as seen by increase in expression of FOXP3 and CD25 in these cells. PT101 maintained selectivity in Phase 1 a clinical trial with no evidence of expansion of natural killer cells nor pro-inflammatory conventional T at any dose studied. A Phase 1b/2a clinical trial in patients with ulcerative colitis and a Phase 2 clinical trial in patients with systemic lupus erythematosus are planned to further evaluate PT101.Disclosure of Interests:None declared

Regulatory T cells (Treg) are vital for immune homeostasis and are dysfunctional in autoimmunity. Interleukin 2 (IL-2) drives the proliferation and function of Tregs via its heterotrimeric receptor (CD25/CD122/CD132). Low dose IL-2 is being evaluated for treatment of autoimmune diseases and has been shown to expand Tregs, yet it has a small selectivity window over conventional T cells (Tconv) and natural killer (NK) cells. To enhance IL-2 selectivity, mutations can be introduced to reduce its CD122/CD132 affinity thus creating a CD25 dependency for signaling through CD122/CD132 upon IL-2 facilitated CD25/CD122/CD132 trimer formation. Using structure guided approach, we introduced mutations in IL-2 that significantly decreased CD122 affinity in addition to mutations that increased CD25 affinity. Finally, we explored other mutations, orientation, and linkers to generate a potent, selective molecule with drug-like manufacturability. These structure activity relationship efforts culminated in the generation of PT101, a mutant IL-2 Fc fusion that was selective in activating and expanding Tregs. PT101 selectively induced STAT5 phosphorylation in human and non-human primate (NHP) Tregs in vitro. In humanized mice and NHPs, administration of PT101 dose-dependently and selectively expanded Treg without significant effects on other immune cell types, nor eliciting proinflammatory cytokine production. In a Phase 1a clinical trial, PT101 was well-tolerated and selectively expanded total Tregs by up to a mean maximum of 3.6-fold. There was no evidence of expansion of NK cells nor pro-inflammatory Tconv at any dose studied. Clinical trials in patients with ulcerative colitis and systemic lupus erythematosus are planned with PT101.