ABSTRACTRegulatory T cells (Treg) play a critical role in immune homeostasis and

are dysfunctional in many autoimmune diseases. Interleukin 2 (IL-2) via the heterotrimeric IL-2 receptor (IL-2R) drives the proliferation and function of Treg. IL-2Rα/CD25 loss-of-function in mice is associated with Tregdeficiency and widespread autoimmunity. Low dose IL-2 expands Treg and is being evaluated as a therapy for patients with autoimmune and inflammatory diseases. However, IL-2 can also activate other immune cells including conventional T cells and natural killer (NK) cells which express IL-2Rβ/CD122 and IL-2γ/CD132. To enhance IL-2 selectivity for Treg, mutations can be introduced that increase the affinity for CD25 and decrease affinity for CD122/CD132. IL-2 muteins (IL-2M) with these properties are able to selectively activate and expand Treg. Here we describe the activity of PT101, an IL-2M cytokine Fc fusion protein that selectively induces STAT5 phosphorylation downstream of the IL-2R in human and cynomolgus monkey Treg ex vivo. In humanized NOD-scidIL2Rγ-null (NSG) mice, PT101 expands Treg without significant effects on other immune cell types and without inducing pro-inflammatory cytokines. Treg from PT101-dosed humanized mice have increased expression of FOXP3 and CD25, and demethylation at the FOXP3 TSDR and CTLA-4 exon 2 suggesting enhanced function and stability. In cynomolgus monkeys, single dose administration of PT101 dose-dependently and selectively expands Treg.

INTRODUCTIONIL-2 was first discovered and characterized as a molecule that promotes the growth, survival, and proliferation of T cells and natural killer (NK) cells (1). The broader role of IL-2 in regulating immune homeostasis was not appreciated until mice deficient in either IL-2 or IL-2Rα/CD25 were generated (2, 3). These animals exhibited widespread autoimmunity and inflammation, which led to the discovery of a subpopulation of IL-2-dependent, CD25-expressing T cells with regulatory function, Treg.IL-2 binds to cells through the IL-2R, which is comprised of IL-2Rα/CD25, IL-2Rβ/CD122, and IL-2Rγ/CD132. CD122 and CD132 expression is sufficient for IL-2 binding and signal transduction (4), whereas CD25 is unable to transduces signals by itself. The heterotrimeric IL-2R including all 3 subunits has high affinity for IL-2 and enables IL-2-dependent signal transduction at IL-2 levels that are insufficient to stimulate through the CD122 and CD132 heterodimer (5). Resting conventional T cells (Tconv) and NK cells express low levels of CD25 until stimulated, whereas Tregconstitutively express high levels of CD25. Treg are therefore responsive to levels of IL-2 that are inadequate to stimulate Tconv and NK cells (6). IL-2M are recombinant forms of IL-2 with amino acid substitutions designed to alter binding of IL-2 to the different IL-2R subunits. Reducing, but not abolishing, the affinity of IL-2M for CD122 biases IL-2-mediated activation away from cells expressing only CD122/CD132 and towards cells that also express CD25. Autoimmune and inflammatory diseases result from disrupted immune homeostasis and inappropriate immune activity, often driven by effector T cells. Treg attenuate inflammatory processes by a variety of mechanisms that suppress the activity of effector T cells (6). Expansion of Treg using repeated administration of low doses of IL-2 has shown preliminary evidence of efficacy in small proof-of-concept clinical trials in patients with a variety of autoimmune diseases (7-13).

RESULTS

Figure 1: Generation of PT101, an IL-2 mutein. IL-2 asparagine 88 is in close contact with residues in IL-2Rβ/CD122 (14). Substitutions at N88 in IL-2 disrupt the H-bond with Q70 in IL-2Rβ, thus reducing its affinity. A substitution at N88 and additional mutations were introduced to generate the IL-2M Fc fusion protein, PT101.

RESULTS

RESULTS

SUMMARYHere we report the development of a novel IL-2M, PT101, that selectively

activates and expands Treg without affecting other cell types. Ex vivo, PT101 selectively induces STAT5 phosphorylation in human and Cyno Treg but not Tconvor NK cells. In humanized NSG mice, PT101 selectively activates and expands Treg in a dose-dependent fashion and with a peak effect at day 7 after a single dose. In vivo-expanded Treg are characterized by increased CD25 and FOXP3 expression and demethylation at the FOXP3 TSDR and CTLA-4 exon 2 suggesting increased function and stability. PT101 treatment does not increase the levels of inflammatory cytokines further indicating selective activation of Treg over other immune cell types. In cynomolgus monkeys, PT101 selectively expands Tregversus other immune cells dose dependently and with a peak effect at day 7. Thereafter, the absolute number of Treg approaches but still remains elevated about baseline levels. Pandion Therapeutics is developing PT101 as a therapeutic approach for the treatment of a variety of autoimmune and inflammatory diseases.

Selective Expansion of Regulatory T cells by IL-2 Muteins Erik R. Sampson1, Jyothsna Visweswaraiah1, Patrick Halvey1, Nathan Higginson-Scott1, Joanne L. Viney1, Kevin L. Otipoby1

Pandion Therapeutics, Cambridge MA 1

Proprietary Mutations

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cytokine receptor. PNAS. 2006;103(8):2788-93.

Figure 2: PT101 selectively activates human and Cyno Treg ex vivo. Isolated human or Cyno peripheral blood mononuclear cells (PBMCs) were treated with PT101 (Upper) or wild type human IL-2 (Lower), stained with surface and intra-cellular marker detection antibodies and analyzed on an Attune NXT flow cytometer to measure STAT5 phosphorylation in Treg, Tconv and NK cells.

- PT101 - PT101 - PT101

- WT IL-2 - WT IL-2 - WT IL-2

- PT101- PT101- PT101

- PT101- PT101- PT101

Figure 3: PT101 selectively expands and activates Treg in humanized mice.CD34-positive hematopoietic stem cell (HSC) humanized NSG mice were dosed once subcutaneously (SQ) with PT101. One week later, whole blood was collected for flow cytometric analysis of Treg frequency and phenotype (Upper) and selectivity (Lower).

- PT101- PT101- PT101

Figure 4: PT101 does not induce pro-inflammatory cytokines in humanized mice.Plasma from PT101-dosed humanized NSG mice was assayed for cytokine levels using the Mesoscale Discovery proinflammatory panel 1 (human) V-plex kit. PT101 does not increase interferon-γ or IL-8 levels and increases IL-10 levels. Other cytokines measured were below the lower limit of quantitation (LLOQ).

Figure 5: Selective expansion of Treg in PT101-dosed mice peaks at Day 7.At the indicated time points following a single SQ dose of PT101, whole blood was collected and analyzed for immune cell frequencies (Upper) and CD25 levels (Lower).

Figure 6: Treg from PT101-dosed humanized mice exhibit demethylation at the FOXP3 TSDR and CTLA-4 exon 2.Sorted Treg or Tconv from PT101 mice humanized with HSCs from two different donors were analyzed for demethylation of the FOXP3 Treg-specific demethylated region (TSDR) in intron 1 (Left) or CTLA4 (Right) by pyro-sequencing or targeted next generation sequencing, respectively.

Figure 7: PT101 selectively expands Treg in cynomolgus monkeys.Whole blood from Cynomolgus monkeys given a single SQ dose of PT101 was stained with surface and intracellular marker antibodies and analyzed by flow cytometry to determine the frequencies of Treg in a dose response study at day 7 (Left), the absolute numbers of Treg over 28 days (Middle) and the fold change in the absolute numbers of Treg versus other immune cells at day 7 (Right).

PDB ID code 2ERJ