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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

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

Human CD7 is an Ig superfamily molecule that is expressed on mature T and NK lymphocytes. Although in vitro studies have suggested a role for CD7 in lymphoid development and function, the exact function of CD7 in vivo has remained elusive. One patient has been reported with SCID syndrome attributed to CD7 deficiency. To study in vivo functions of CD7, we have generated CD7-deficient mice and assessed their lymphoid development and function. CD7-deficient mice were viable, had normal peripheral blood and spleen lymphocyte numbers, and had normal specific Ab responses with Ag-driven Ig isotype switching. Thymocyte numbers were normal in 4-wk-old, 6-mo-old, and 1-yr-old CD7-deficient mice, but in 3-mo-old CD7-deficient mice, total thymocyte numbers were significantly increased by 60% (p0.02) compared with normal age-matched +/+ littermates. CD7-deficient splenocytes proliferated normally in response to various mitogens, including PHA, anti-CD3, Con A, and LPS. While NK cell numbers and cytolytic activity to YAC targets were normal, CD7-deficient mice had lower Ag-induced MHC class I-restricted CTL activity against OVA-transfected target cells than did +/+ control mice. Thus, CD7-deficient mice did not have a SCID syndrome, but rather had transient increases in thymocyte numbers at age 3 mo and altered splenocyte Ag-specific CTL effecter cell activity. These data suggest a role for CD7 in regulating intrathymic T cell development and in mediating CTL effecter function.

Toxicity limits the use of methotrexate.To identify risk factors for methotrexate-induced lung injury in patients with rheumatoid arthritis.Case-control study.One private and five academic rheumatology practices.Methotrexate recipients with rheumatoid arthritis with and without lung injury.Potential risk factors examined were sociodemographic and lifestyle characteristics, medical history, clinical and ancillary features and treatment of rheumatoid arthritis before methotrexate therapy, and characteristics of methotrexate therapy. Cases of lung injury were defined according to the modified criteria of Searles and McKendry.Ninety-four percent of the study participants were white, and 67% were women. Case-patients (n = 29) were older than controls (n = 82) (61.5 compared with 54.5 years of age). The strongest predictors of lung injury, after adjustment for other variables, were older age (odds ratio [OR], 5.1 [95% CI, 1.2 to 21.1]), diabetes (OR, 35.6 [CI, 1.3 to infinity]), rheumatoid pleuropulmonary involvement (OR, 7.1 [CI, 1.1 to 45.4]), previous use of disease-modifying antirheumatic drugs (OR, 5.6 [CI, 1.2 to 27.0]), and hypoalbuminemia (OR, 19.5 [CI, 3.5 to 109.7]). Previous use of disease-modifying antirheumatic drugs and hypoalbuminemia had very large attributable risks.Knowledge of the risk factors that predispose patients with rheumatoid arthritis to the toxic effects of methotrexate on the lung may provide a rationale for monitoring high-risk patients and may facilitate their management.

The pathogenesis of vasculitis syndromes is via a diverse array of mechanisms that disrupt vessel integrity and produce clinical signs and symptoms of tissue ischemia. In many patients with vasculitis, it is not possible to directly classify the syndrome according to a particular schema, and attempts to define the pathogenic process mediating vessel damage by biopsy and laboratory tests may be useful to guide therapy. The continued use of new techniques to probe immune cell types and cytokines in vasculitis lesional tissues will further clarify molecular mechanisms of vascular damage, and it is hoped that they will lead to new and more effective therapeutic strategies targeted at the vasculitis syndromes.

T cell epitopes residing within vaccine candidate peptides have been identified by delayed-type hypersensitivity (DTH) responses in mice. The recombinant sporozoite vaccine candidate, R32tet32, contains at least two T epitopes, one located within the repeat region and another in the tet tail. When C57BL/6 (H-2b) and BALB/c (H-2d) mice were sensitized intradermally with R32tet32 or the truncated protein R32LR emulsified in CFA and challenged 5 days later with R32tet32, only H-2b mice recognized a T epitope located within the major repeat sequence (NANP) and encoded by four or less repeats. H-2d mice responded solely to the T epitope located on the tet tail. Ear swelling was maximal at 48 h and revealed a histologic pattern characteristic of DTH. CD4+ T cell lines derived from immunized animals demonstrated the ability to mediate local DTH, proliferate, and secrete lymphokines in response to stimulation with Ag. High dose i.v. administration of R32tet32 in C57BL/6 and BALB/c mice before intradermal sensitization with R32tet32 revealed that DTH responses were suppressed only in BALB/c mice. Further experiments localized the suppressive determinant to the tet tail. Collectively, these data indicate that DTH may prove to be a useful method to characterize the biologic activity of T epitopes, furthermore they suggest that candidate vaccine peptides should be tested for suppressive activity before inclusion in a vaccine.