Selective Treg Research Articles

Human OX40L-CAR-Tregs target activated antigen-presenting cells and control T cell alloreactivity.

Regulatory T cells (Tregs) make major contributions to immune homeostasis. Because Treg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through Treg adoptive transfer. Two of the central challenges in clinically deploying Treg cellular therapies are ensuring phenotypic stability and maximizing potency. Here, we describe an approach to address both issues through the creation of OX40 ligand (OX40L)-specific chimeric antigen receptor (CAR)-Tregs under the control of a synthetic forkhead box P3 (FOXP3) promoter. The creation of these CAR-Tregs enabled selective Treg stimulation by engagement of OX40L, a key activation antigen in alloimmunity, including both graft-versus-host disease and solid organ transplant rejection, and autoimmunity, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. We demonstrated that OX40L-CAR-Tregs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of Treg suppressive proteins without induction of proinflammatory cytokine production. Compared with control Tregs, OX40L-CAR-Tregs more potently suppressed alloreactive T cell proliferation in vitro and were directly inhibitory toward activated monocyte-derived dendritic cells (DCs). We identified trogocytosis as one of the central mechanisms by which these CAR-Tregs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L-CAR-Tregs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control Tregs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L-CAR-Tregs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases.

A CD25×TIGIT bispecific antibody induces anti-tumor activity through selective intratumoral Treg cell depletion

Intratumoral regulatory T cells (Tregs) express high levels of CD25 and TIGIT, which are also recognized as markers of effector T cell (Teff) activation. Targeting these molecules each alone with monoclonal antibodies (mAbs) poses a risk of concurrently depleting both Teffs and peripheral Tregs, thereby compromising the effectiveness and selectivity of intratumoral Treg depletion. Here, leveraging the increased abundance of CD25+ TIGIT+ double positive Tregs in the solid tumor microenvironment (but not in peripheral tissues), we explored the feasibility of using a CD25×TIGIT bispecific antibody (bsAb) to selectively deplete intratumoral Tregs. We initially constructed a bsAb co-targeting mouse CD25 and TIGIT, NSWm7210, and found that NSWm7210 conferred enhanced intratumoral Treg depletion, Teff activation, and tumor suppression as compared to the parental monotherapies in mouse models. We subsequently constructed a bsAb co-targeting human CD25 and TIGIT (NSWh7216), which preferentially eliminated CD25+ TIGIT+ double positive cells over single positive cells in vitro. NSWh7216 exhibited enhanced anti-tumor activity without toxicity of peripheral Tregs in CD25 humanized mice compared to the parental monotherapies. Our study illustrates the use of CD25×TIGIT bsAbs as effective agents against solid tumors based on selective depletion of intratumoral Tregs.

406.7: Inducing long-term heart allograft tolerance through selective Treg expansion by IL-2 complex treatment.

406.7: Inducing long-term heart allograft tolerance through selective Treg expansion by IL-2 complex treatment.

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

S-531011, a Novel Anti-Human CCR8 Antibody, Induces Potent Antitumor Responses through Depletion of Tumor-Infiltrating CCR8-Expressing Regulatory T Cells.

Although regulatory T cells (Treg) are inhibitory immune cells that are essential for maintaining immune homeostasis, Tregs that infiltrate tumor tissue promote tumor growth by suppressing antitumor immunity. Selective reduction of tumor-infiltrating Tregs is, therefore, expected to activate antitumor immunity without affecting immune homeostasis. We previously reported that selective Treg depletion targeted by a C-C motif chemokine receptor 8 (CCR8) resulted in induction of strong antitumor immunity without any obvious autoimmunity in mouse models. Thus, herein, we developed a novel humanized anti-CCR8 monoclonal antibody, S-531011, aimed as a cancer immunotherapy strategy for patients with cancer. S-531011 exclusively recognized human CCR8 among all chemokine receptors and showed potent antibody-dependent cell-mediated cytotoxicity activity toward CCR8+ cells and neutralization activity against CCR8-mediated signaling. We observed that S-531011 reduced tumor-infiltrating CCR8+ Tregs and induced potent antitumor activity in a tumor-bearing human-CCR8 knock-in mouse model. Moreover, combination therapy with S-531011 and anti-mouse programmed cell death 1 (PD-1) antibody strongly suppressed tumor growth compared with anti-PD-1 antibody alone with no observable adverse effects. S-531011 also depleted human tumor-infiltrating Tregs, but not Tregs derived from human peripheral blood mononuclear cells. These results suggest that S-531011 is a promising drug for inducing antitumor immunity without severe side effects in the clinical setting.

NEO-TRA1: A CD25-Targeted De Novo non-Alpha Agonist of the IL-2 Receptor Selectively Expands Regulatory T Cells

Introduction: Regulatory T cells (Tregs) are crucial for maintaining immune tolerance and their dysfunction contributes to inflammatory and autoimmune conditions, such as graft versus host disease (GVHD). IL-2 is a pleiotropic cytokine that activates both immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Although low dose IL-2 therapy effectively expands Tregs, this intervention is limited by coincident stimulation of conventional T cells and natural killer cells. Investigational therapeutics derived from directed mutagenesis of native IL-2 sequences have resulted in some improvements in Treg selectivity. To optimize the magnitude and selectivity of Treg expansion, we utilized de novo protein design technology to fine-tune the individual receptor interfaces of IL-2 mimetic proteins targeted to Tregs with CD25 antibodies. We found decoupling receptor agonism and targeting is an effective means of improving Treg stimulation and corresponding therapeutic candidates elicit robust expansion of Treg populations in vivo without detectable stimulation of inflammatory immune cells. Methods: We designed a series of de novo IL-2Ra independent agonists of the IL-2 receptor that demonstrate reduced binding affinity to CD122 and CD132, the beta and gamma chains of the IL-2 receptor complex, by up to 5 logs when compared to IL-2. These non-alpha IL-2 mimetic variants were fused to a humanized anti-CD25 mAb that lacks effector function for cis-targeting to Tregs. Candidate molecules were screened on primary human T cells to assess pSTAT5 signaling. Treg-specific molecules were administered to humanized NSG mice weekly (four times) and peripheral blood was collected for immune cell profiling via flow cytometry. Results: Screening of TRA candidates in primary human T cells identified several highly selective Treg agonists, including NEO-TRA1 (Fig1). At all concentrations, CD8+ and CD4+ non-Tregs showed low or negligible levels of pSTAT5 signaling upon treatment with NEO-TRA1. Nevertheless, CD4+CD25highCD127low Tregs show increasing pSTAT5 signaling at a wide range of concentrations starting at 20pM, and demonstrate preferential signaling in Tregs by NEO-TRA1. To establish the ability of our TRA to expand Treg in vivo, NSG mice engrafted with human hematopoietic stem cells to create a humanized immune system received weekly injections of NEO-TRA1, vehicle control, or a previously described Treg selective IL-2 mutein (IL-2v-Fc). Characterization of Day 13 CD4+CD25+Foxp3+ Treg percentages in peripheral blood mononuclear cells show that the Treg population expanded to ~30% of total CD4+ cells in mice receiving NEO-TRA1, while Treg percentages reached ~22% in mice receiving IL-2v-Fc (Fig2). Despite a slight decrease at Day 20, the Treg percentages remained higher in NEO-TRA1-treated cohorts than in control groups throughout the study. Conclusions: NEO-TRA1 exhibits potent expansion of Treg populations within primary human samples and in humanized mouse models. The observed profile of STAT5 phosphorylation demonstrates selective activation of Tregs over other lymphocyte populations. NEO-TRA1 is the first Treg agonist based on de novo protein design. NEO-TRA1 is both specific and potent and has high therapeutic potential for the treatment of GVHD and other autoimmune or inflammation diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AB0432 EFAVALEUKIN ALFA, A NOVEL IL-2 MUTEIN, SELECTIVELY EXPANDS REGULATORY T CELLS IN PATIENTS WITH SLE: FINAL RESULTS OF A PHASE 1B MULTIPLE ASCENDING DOSE STUDY

BackgroundDefects in regulatory T cell (Treg) number and function are associated with autoimmune diseases including SLE. Interleukin (IL)-2 is essential for the development and suppressive function of Treg, and therapies that exploit the ability of IL-2 to expand Treg have shown disease-modifying potential in SLE1. However, low-dose IL-2 has a short half-life and narrow selectivity for Treg over conventional CD4+ T cells (Tcon) and natural killer (NK) cells. Efavaleukin alfa is an IL-2 mutein Fc fusion protein; an introduced mutation decreases binding to IL-2Rβ and increases dependence on IL-2Rα (CD25). This preferential binding to the high-affinity IL-2R, constitutively expressed at high levels on Treg, leads to increased cell surface retention and sustained Treg signaling compared with recombinant IL-2. In healthy subjects, a single dose of efavaleukin alfa was well tolerated and led to robust and selective Treg expansion2.ObjectivesThis final analysis of a phase 1b, double-blind, placebo-controlled, multiple ascending dose study (NCT03451422) reports the safety, tolerability, pharmacokinetics (PK), and pharmacodynamic effects of efavaleukin alfa in patients with SLE.MethodsThe study included five ascending dose cohorts (cohort 1=lowest dose; cohort 5=highest dose). A total of 35 patients with SLE (age 24–71 years; 85.7% female; SLE diagnosed using SLICC or ACR criteria with ANA ≥1:80 and/or elevated anti-dsDNA antibodies) were randomized to receive efavaleukin alfa or placebo (5:2 ratio for cohorts 1–3; 3:1 ratio for cohorts 4–5) subcutaneously every 2 weeks (Q2W; cohorts 1, 2, 4, and 5) or every week (QW; cohort 3) in addition to standard of care therapy for a total of 12 weeks, with 6 weeks of follow-up. The primary endpoint was the incidence of treatment-emergent adverse events (TEAEs). Additional endpoints included serum PK of efavaleukin alfa and changes in numbers of Treg, CD4+ Tcon, CD8+ T cells, and NK cells in peripheral blood.ResultsThe most commonly reported TEAEs (occurring in ≥25% of efavaleukin alfa-treated subjects) included non-serious, mild or moderate (grade 1–2) injection site reactions. No grade 4 TEAEs or deaths occurred. Two serious AEs were reported in efavaleukin alfa-treated subjects: one event of syncope (grade 3) was observed in cohort 2 and was not considered related to treatment, and one case of eosinophilia (grade 2) was observed in cohort 5 and was considered related to treatment. Efavaleukin alfa PK was generally linear and dose-proportional, with a terminal half-life ranging from 18–30 hours. Peak Foxp3+ Treg expansion was observed at 8 days post-dose, and the magnitude of the peak was generally sustained after multiple QW or Q2W doses. The mean peak increases in Foxp3+ Treg were 14.8-, 17.4-, 5.7-, 2.4-, and 1.1-fold above baseline for efavaleukin alfa Q2W dosing cohorts 5, 4, 2, and 1 and placebo, respectively. At the final study assessment (42 days after the last dose), the mean Treg count was 1.3-fold above baseline (95% CI, 0.9–1.9). Treatment with efavaleukin alfa also expanded CD25bright Treg (peak 53.8-fold change) and CD31+ recent thymic emigrant (RTE), naïve, and memory Treg subsets. At the highest dose (cohort 5), low-level increases in numbers of CD4+ Tcon (peak 2.3-fold), CD8+ T cells (peak 2.1-fold), and NK cells (peak 2.9-fold) were observed.ConclusionMultiple ascending doses of efavaleukin alfa were safe and well tolerated and led to selective and prolonged Treg expansion in SLE patients. Results at the highest dose suggest a plateau in Treg expansion with low-level increases in other IL-2–responsive cells, although interpretation is limited due to small subject numbers. The highest tested dose may be outside the therapeutic window and thus will not be assessed in phase 2 clinical studies. These findings confirm and extend previous results in healthy subjects and support the ongoing phase 2b adaptive randomized controlled trial in patients with SLE.

Abstract 1847: Anti-GARP antibody DS-1055a augments antitumor immunity by depleting highly suppressive GARP+ regulatory T cells

Abstract Immune checkpoint blockers (ICBs) have drastically changed the clinical care of cancer; however, the population of patients who can benefit is relatively small because of intrinsic or acquired resistance to immune therapy. To evade immune destruction, tumors exploit several distinct strategies including immunosuppressive cells such as regulatory T (Treg) cells. Treg cells, an essential component for maintaining self-tolerance, inhibit antitumor immunity, consequently hindering protective cancer immunosurveillance and hampering effective antitumor immune responses in tumor-bearing hosts. It is often reported that a high ratio of Treg cells to effector CD8+ T cells is associated with poor prognosis in patients with cancer, and Treg cells represent one of the most important factors associated with resistance to ICBs, suggesting that Treg cells represent a new promising target for anti-cancer therapy. In this study, we performed a comprehensive screening to identify a tumor-infiltrating Treg cell-specific molecule among immune cell subsets in humans. Glycoprotein A repetitions predominant (GARP) was identified as a suitable target for selective Treg cell depletion in the tumor microenvironment (TME) of multiple caner types. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation in humans. DS-1055a, a novel afucosylated anti-human GARP antibody, effectively depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FOXP3+CD4+ T cell counts in the TME and exhibited remarkable antitumor activity in a human peripheral blood mononuclear cell-transplanted humanized mouse model bearing HT-29 tumors. From these results, we propose that DS-1055a can be a new Treg cell-targeted regent that augments antitumor T cell immunity by depleting GARP+ Treg cells. Currently, a Phase I study of DS-1055a is on-going (NCT04419532). Citation Format: Kazuki Satoh, Yoichi Kobayashi, Kaori Fujimaki, Masato Hata, Shinko Hayashi, Saori Ishida, Daisuke Sugiyama, Takahiko Sato, Lim Kyungtaek, Megumi Miyamoto, Shiho Kozuma, Michinori Kadokura, Kenichi Wakita, Kazuki Hirahara, Masato Amano, Ichiro Watanabe, Atsushi Okamoto, Andrea Tuettenberg, Helmut Jonuleit, Atsushi Tanemura, Shoichi Maruyama, Toshinori Agatsuma, Teiji Wada, Hiroyoshi Nishikawa. Anti-GARP antibody DS-1055a augments antitumor immunity by depleting highly suppressive GARP+ regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1847.

Abstract 1677: Characterization of the pharmacodynamic activity of AGEN1181, an Fc-enhanced CTLA-4 antibody, alone and in combination with the PD-1 antibody balstilimab

Abstract AGEN1181 is a novel, Fc-engineered monoclonal antibody that targets cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), with enhanced FcyR-dependent functionality designed to improve T cell priming and activation, deplete intratumoral regulatory T cells (Treg), and improve memory T cell response. AGEN1181 is currently undergoing Phase I clinical evaluation, both as a single agent and in combination with the anti-PD-1 antibody balstilimab, in patients with advanced solid tumors (NCT03860272). Encouraging signals of clinical benefit have emerged as part of this ongoing study, including complete and durable responses in patients that would traditionally be considered unresponsive to conventional CTLA-4/PD-1 therapy. Here we report on pharmacodynamic analyses associated with AGEN1181 exposure in patients on study in this trial. As monotherapy, AGEN1181 was administered on a once every 3 or once every 6 week cycle (doses escalated from 0.1 - 4 mg/kg). Alternatively, AGEN1181 was dosed once every six weeks in combination with balstilimab (3 mg/kg once every 2 weeks). Tumor mutational burden (TMB) and gene expression were assessed from pre-treatment and on-treatment biopsies by whole exome sequencing (WES) and RNASeq, respectively. Multiplex immunohistochemistry was used to assess Treg depletion and CD8 T-cell activation in response to AGEN1181. TCR repertoire was analyzed in PBMCs collected on-treatment with AGEN1181. FcγRIIIA genotyping was performed by real-time PCR. Immunophenotyping of peripheral blood mononuclear cells collected pre- and post-treatment were analyzed by flow cytometry. Consistent with its proposed mechanism of action, dose-dependent pharmacodynamic effects have been observed in patients while on-treatment with AGEN1181. Peripheral CD4+Ki67+, CD4+ICOS+ and CD4+HLA-DR+ T cells increased over baseline in response to monotherapy. This effect was further enhanced in subjects treated with AGEN1181 when given in combination with balstilimab. Responders expressed either the low affinity FcγRIIIA allele or were heterozygous - suggesting that AGEN1181 has the potential to expand clinical activity to patients with low affinity Fc binding, in contrast to conventional CTLA-4 inhibitors. Additionally, we show that AGEN1181 induced selective Treg depletion within the tumor microenvironment with concomitant increases in CD8 T cell infiltration. TCRSeq analysis demonstrated increased clonality and decreased downsampled diversity, providing further evidence that the Fc-enhanced activity of AGEN1181 can promote improved T cell priming and Treg depletion. In summary, the pharmacodynamic analyses presented here are consistent with the Fc-enhanced design of AGEN1181 and its potential to broaden the therapeutic reach of CTLA-4-based immunotherapy. Citation Format: Irina Shapiro, Min Lim, Simarjot Pabla, Steven J. O'Day, Anthony El-Khoueiry, Chethan Ramamurthy, Andrea J. Bullock, Michael S. Gordon, Jennifer Buell, Dhan Chand, Anna Wijatyk. Characterization of the pharmacodynamic activity of AGEN1181, an Fc-enhanced CTLA-4 antibody, alone and in combination with the PD-1 antibody balstilimab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1677.

Individual Patient Dose-Escalated Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease in Children and Adults: Safety, Efficacy and Immune Correlates

CD4+CD25+FoxP3+ regulatory T cells (Treg) are broadly suppressive and play a central role in the prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic cell transplantation (HCT). Daily subcutaneous (SC) low-dose interleukin-2 (IL-2) at a fixed dose of 1x106 IU/m2/day in adult patients with refractory cGVHD induces selective Treg expansion and leads to clinical responses in 50-60% of evaluable participants (NEJM 2011, Blood 2016). During daily fixed-dose IL-2 therapy, plasma IL-2 levels rose rapidly by week 1, but subsequently decreased over time. The lower IL-2 levels were associated with a decline in Treg proliferation despite daily IL-2 administration (Sci Transl Med 2013), possibly due to IL-2 sequestration via binding to constitutively-expressed high affinity IL-2 receptors on Treg. We reasoned that IL-2 dose escalation at the time of anticipated fall in plasma IL-2 levels would avoid tachyphylaxis and maximize IL-2 induced Treg numbers in vivo.We conducted a phase 1 trial of individual patient IL-2 dose escalation over 8 weeks in adult and pediatric patients with steroid-refractory cGVHD. Daily SC IL-2 was initiated at 0.67x106 and 0.33x106 IU/m2/day in the adult and pediatric cohorts, respectively. Each participant had dose escalations at weeks 2 and 4 to a maximum dose of 2x106 IU/m2/day in adults and 1x106 IU/m2/day in children. We studied 18 HCT recipients (10 adult: 100% HLA-matched, 100% PBSC grafts, 40% prior aGVHD; 8 pediatric: 87.5% HLA-matched, 87.5% BM grafts, 28.6% prior aGVHD). Median age was 57 years (range, 33-71) in the adult cohort and 12 years (range, 5-22) in the pediatric cohort. The median time from HCT and from cGVHD onset to start of IL-2 treatment was 3 years (range, 1-12.1) and 597 days (range, 234-4336) respectively. Participants had a median of 2 cGVHD organ sites (range, 1-5) and 3 prior cGVHD therapies (range, 1-4) at enrollment. The cohorts did not differ in terms of time since cGVHD onset and number of failed therapies prior to enrollment.Individually dose-escalated IL-2 was well tolerated in children with 1 participant requiring dose reduction for electrolyte imbalances. In the adult cohort, 1 participant withdrew due to cGVHD progression, 1 required dose reduction for injection site reactions and 2 were unevaluable (1 IL-2 hold >1 month, 1 non-compliance). Seven grade 3 AEs were reported for six participants (electrolyte imbalance, vomiting, infection, increased ALT and soft tissue neoplasm); none relapsed. At week 8, objective cGVHD responses (PR) were seen in 7 of 8 pediatric participants (88%), but in only 1 of 8 evaluable adult participants (13%). cGVHD response sites included skin (n=4), joint/fascia/muscle (n=1), lung (n=4), and GI tract (n=2). Eight pediatric and 4 adult participants with clinical benefit (PR or SD with minor response) continued extended duration IL-2 therapy up to 120 weeks.Plasma IL-2 levels were higher in adults at weeks 1 and 2, but immune responses were similar between the 2 cohorts. Dose escalated IL-2 induced a >10-fold increase in median Treg count/µL that peaked at week 6. There were no significant changes in CD4 Tcon, CD8 T cell or B cell count. NK cell count increased >5-fold by week 8. The median Treg:Tcon ratio increased 6-fold from a baseline of 0.05 (Q1-Q3, 0.024-0.114) to a week 5 peak of 0.312 (Q1-Q3, 0.203-0.37). Compared with fixed daily dosing of IL-2 (Blood 2016), the individually dose escalated IL-2 schedule had a slower initial rise in Treg number and Treg:Tcon ratio (Figure 1). This likely reflects the lower starting doses in this study. The maximum IL-2 dose in the adults was twice the previously defined MTD but did not improve Treg expansion. Despite lower IL-2 doses, pediatric patients had a superior clinical response and a trend toward a better immunologic response compared with the adults.We demonstrate for the first time that low-dose IL-2 is safe in children with advanced steroid-refractory cGVHD and results in a high clinical response rate. A maximum dose of 1x106 IU/m2/day was well tolerated by pediatric patients in the extended duration phase and durably maintained Treg numbers above baseline. Five pediatric patients that continued IL-2 therapy for >20 weeks have reduced or discontinued steroid therapy. In adults, intrapatient dose escalation above fixed-dose MTD was not clinically tolerated and did not result in greater Treg expansion or clinical improvement relative to fixed daily dosing. [Display omitted] DisclosuresNikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Roche: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Infinity: Consultancy; Genmab: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Tensha: Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Koreth:Prometheus Labs: Research Funding; Amgen, Inc: Consultancy.

A Crohn's Disease-associated IL2RA Enhancer Variant Determines the Balance of T Cell Immunity by Regulating Responsiveness to IL-2 Signalling.

Background and AimsDifferential responsiveness to interleukin [IL]-2 between effector CD4+ T cells [Teff] and regulatory T cells [Treg] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn’s disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP.MethodsTeff and Treg were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. Teff pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation.ResultsPresence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by Teff in response to IL-2 stimulation in vitro. Teff from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response.Conclusionsrs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective Treg modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype.

Mobilized Multipotent Hematopoietic Progenitors Promote Expansion and Survival of Allogeneic Tregs and Protect Against Graft Versus Host Disease.

Allogeneic Hematopoietic Stem Cell Transplantation (Allo-HSCT) is routinely performed with peripheral blood stem cells (PBSCs) mobilized by injection of G-CSF, a growth factor which not only modulates normal hematopoiesis but also induces diverse immature regulatory cells. Based on our previous evidence that G-CSF-mobilized multipotent hematopoietic progenitors (MPP) can increase survival and proliferation of natural regulatory T cells (Tregs) in autoimmune disorders, we addressed the question how these cells come into play in mice and humans in an alloimmune setting. Using a C57BL/6 mouse model, we demonstrate that mobilized MPP enhance the immunosuppressant effect exerted by Tregs, against alloreactive T lymphocytes, both in vitro and in vivo. They do so by migrating to sites of allopriming, interacting with donor Tregs and increasing their numbers, thus reducing the lethality of graft-versus-host disease (GVHD). Protection correlates likewise with increased allospecific Treg counts. Furthermore, we provide evidence for a phenotypically similar MPP population in humans, where it shares the capacity to promote selective Treg expansion in vitro. We postulate that G-CSF-mobilized MPPs might become a valuable cellular therapy to expand donor Tregs in vivo and prevent GVHD, thereby making allo-HSCT safer for the treatment of leukemia patients.

Abstract PO082: Significance of Treg cells in pathogenesis of oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (OSCC) is one of the major cancers affecting in Asian countries. The main causative factor has been tobacco habit. It has been reported that immune dysfunction in these patients is one of the major factors for tumor growth and dissemination that affects disease free survival of the patients. We assessed the phenotypic and functional characteristics of Regulatory T (Treg) CD4+CD25+FoxP3+subsets in patients with OSCC by multicoloured flow cytometry. Subsequently we investigated the effects their inhibition via TDG on growth of OSCC cell lines in vitro. An increased (p<0.05) prevalence of Treg phenotypes (CD4+CD25+, CD4+FoxP3+, CD8+FoxP3+, CD4+CD25+FoxP3+) was observed in the peripheral circulation of OSCC patients that positively correlated with clinicopathological features. The increased frequency of CD4+CD8+CD25+FoxP3+, a unique T cell subset, CTLA4+, GITR+, NrP1+ and granzyme B+ (GzmB) Tregs also showed a significantly higher prevalence in OSCC patients. Functionally CD4+FoxP3+ Tregs showed skewed expression of IL2, IL10 and IL35 in patients as compared with the normal controls. Higher expression of TGFβ in tumor tissues suggests their dominant role in the up regulation of differentiation of Tregs from naive T cells in the tumor bearing host. Further, enhanced expression of CCR5 and CCR7 on Tregs with up regulation of their ligands (CCL5, CCL19 and CCL21) in tumor cells indicates efficient recruitment and trafficking of Tregs to the tumor site. Gal-1 expression was observed to be significantly higher in oral cancer patients. Treatment with βGBP showed growth promoting effects on Tregs and oral cancer cells. However, the treatment with its inhibitor TDG resulted in inhibition of Treg subsets and also decreased the frequency of IL10+ and IL35+ Tregs indicating its immunomodulatory effects. Hence, it seems reasonable to assume that modulation of functional dynamics of selective Treg subsets may be useful in enhancing anti tumor immunity and developing immunotherapeutic strategies for patients with oral squamous cell carcinoma. Citation Format: Sadhna Aggarwal, Suresh C. Sharma, Satya N. Das. Significance of Treg cells in pathogenesis of oral squamous cell carcinoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO082.

An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice.

Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (Treg) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved Treg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced Treg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective Treg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall Treg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential Treg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of Treg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules.

Abstract 4553: The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control

Abstract Despite the advances in cancer immunotherapy, in particular in the field of checkpoint inhibitors (CPI), many patients fail to respond (primary resistance) or initially benefit but then progress upon treatment (secondary resistance). High regulatory T-cell (Treg) counts correlate with poor prognosis and reduced responsiveness to CPI therapy in humans, underscoring their potential as an immunotherapy target. Clinical attempts aiming to lower Treg counts, however, either failed to deliver convincing Treg reduction or lacked specificity for Treg over tumor antigen specific cytotoxic T cells (CTL). CD25 (the interleukin-2 receptor alpha (IL-2Ra) chain) is a recently revisited target for Treg depletion. For privileged access to IL-2, activated CTL up-regulate CD25 expression only transiently during clonal expansion. Further confirmation of an > 20 fold higher cell surface expression of CD25 on Tregs versus CTLs in human malignancies is provided. The novel compound RG6292 was developed as an ADCC and ADCP competent monoclonal antibody of human IgG1 isotype with afucosylated glycans in the Fc region. RG6292 binds with low monovalent affinity (KD 250 nM) to the extracellular domain of CD25 antigen. A high density of CD25 receptors promotes bivalent avidity of RG6292 increasing its binding strength to CD25 by at least 100 fold (KD 2-3 nM). RG6292 selectively favors the depletion of CD25 high Tregs over CD25 low activated CTLs, here shown in comparison to ipilimumab and mogamulizumab in human αCD3 activated PBMC, human tumor explants and immunopharmacodynamic studies in tumor bearing (BxPC-3), stem cell humanized mice and cynomolgus monkeys. IL-2 is an essential prerequisite for clonal expansion of CTLs, which is necessary to generate effective anti-tumor responses. Earlier immunosuppressant anti-CD25 antibodies (e.g. daclizumab and basiliximab) interfered with the formation of the high affinity IL-2R complex. Their evidenced lack of therapeutic activity in immunoncology tempered enthusiasm and highlights the pivotal role of IL-2. RG6292 is the first anti-human CD25 antibody developed to deplete Tregs selectively while fully preserving IL-2 signaling and CTL activity. Pre-clinically, a single administration of the RG6292 surrogate effectively promoted eradication of established tumors in several tumor mouse models and synergized with CPI in models of CPI resistance. RG6292 is expected to unleash the potential of selective Treg depletion while allowing for unrestricted access of IL 2 to CTLs and could therefore result in clinically superiority compared to other Treg depleting antibodies. RG6292 provides a novel therapeutic approach to alleviate a major mechanism of immune suppression in the tumor microenvironment. Clinical testing is currently ongoing to evaluate the safety and tolerability of RG6292 in patients with advanced solid tumors (NCT04158583). Citation Format: Maria Amann, Gabriel Schnetzler, Kolben Theresa, Isabelle Solomon, Christophe Boetsch, Estelle Marrer-Berger, Reto Flury, Claudio Murgia, Vaios Karanikas, Johannes Sam, Roger Sutmuller, Jan Eckmann, Hans Koll, Sara Belli, Frederic Arce Vargas, Dimitrios Zervas, Chen Qing, Mark A. Brown, Josephine Salimu, Anne Goubier, Sebastian Neumann, Karl S. Peggs, Sergio A. Quezada. The CD25 antibody RG6292 selectively depletes Tregs while preserving IL-2 signaling and CTL activity for tumor control [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4553.

Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins.

Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both in vitro and humanized mouse in vivo systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function in vivo.

The Location of CD4+FoxP3+ Cells with Regard to CD25 and TNFRSF25 Receptor Signals Matters: Different Requirements for GI and Peripheral Tregs

Regulatory T cells (Tregs) are critical to maintaining immune homeostasis and generating tolerance in the gastrointestinal (GI) tract. GI complications play a prominent role following allogeneic HSCT (aHSCT) and we are interested in manipulating GI Tregs to regulate GVHD. Previously our lab has shown that using a two-pathway strategy of stimulating the TNFRSF25 and CD25 receptors with a TL1A-Ig fusion protein (FP) and IL-2low dose, Treg frequency and numbers in the lymphohematopoietic (LHC) compartment can be markedly increased (Wolf, BBMT 2017; Copsel, BBMT 2018). As a consequence of microbes and food antigens, Treg populations in the GI have a relative highly diverse composition compared to the lymphohematopoietic Treg compartment. This includes, but is not limited to, the presence of a stable and suppressive FoxP3+RORyt+ Treg population. Additionally, the GI tract contains various populations of innate lymphoid cells (ILCs) that interact with Tregs. ILCs express CD25 as well as TNFRSF25, and respond individually to IL-2 and TL1A administration respectively (Danny, JCI 2017; Verneris, Blood 2013). Based on the extensive differences between the lymphohematopoietic and GI Treg compartments, we evaluated how our two-pathway strategy may be differentially affecting the levels and activation status of Tregs in the GI tract. To address this question, we initially utilized B6-Nur77GFP mice, where GFP expression occurs with activation of the Nur77 promoter. However, since Nur77 is only produced following TcR engagement and not inflammatory signals, the strength of TcR stimulation in all T cell populations can be monitored by GFP levels. We first examined Tregs from 2-pathway treated B6-Nur77GFP mice and subsequently generated B6-Nur77GFPFoxP3RFP mice to readily assess the TcR signaling status of Tregs in different compartments. Mice were systemically administered FP, IL-2, or FP + IL-2 over a 1 wk period. The frequency of Tregs (FoxP3+CD4+) / Tcon (CD4+FoxP3-) in the lamina propria (LP) of the large intestine (LI) reached levels >60%. (1A). This elevation of Treg / Tcon frequency included FoxP3+RORyt- Tregs as well as FoxP3+RORyt+ double positive Treg populations. In contrast to our previous findings in the LHC, treatment with TL1A-Ig FP alone elevated levels to the same extent as the combination of FP + IL-2 (1A). Importantly, IL-2 treatment alone - as reported in the LHC - again had only a modest effect on elevating the frequency of Tregs in the LI/LP (1A). These observations suggest that the activation status of Tregs may differ based on the compartmental location. To asses activation status, we evaluated 2-pathway treated B6-Nur77GFP mice. Tregs in the LN/spleen had elevated frequency of GFP+ Tregs and higher GFP MFI than untreated mice. This elevated TcR stimulation was present in peripheral Tregs - but not CD8 - T cells (1B). Without exogenous stimulation, Tregs exhibited higher baseline TcR activation levels vs. Tcon cells (1B). The frequency of GFP+ Tregs and the GFP MFI was clearly highest (>45%) in the SI and LI LP vs. LN/spleen (<35%) (1C). In an independent experiment, we verified these findings and learned that only conjunctival Tregs demonstrated similarly increased frequency of GFP+ Tregs with elevated GFP MFI (1D). We also found that TL1A-Ig + IL-2 treatment in vivo increases the relative frequency of both innate ILC2 and ILC3 lymphoid populations (2A). The combination also increased the frequency of ILC2 cells greater than either reagent alone in the GI tract (2B). In total, our results objectively validate selective Treg vs. Tcon targeting via TNFRSF25. These findings also demonstrate that basal Treg activation status differs depending on the compartment. Notably, Tregs in mucosal vs. LHC tissue expressed higher TcR activation levels. Such Tregs have the potential for co-stimulation via TNFRSF25. Moreover, since IL-2 is required to maintain Tregs under both basal and activated conditions, our findings suggest a local source of IL-2 is present to maintain GI Tregs. ILC3 cells are recently reported to generate IL-2 (Zhou L, Nature 2019). We hypothesize that TNFRSF25 stimulation in ILC3 cells results in a local source of IL-2 which can account for our finding that TL1A-Ig alone vs. TL1A-Ig + IL-2 stimulation results in equivalent levels of GI Tregs. Ongoing experiments are examining how GI GVHD in aHSCT recipients are affected by TNFRSF25 +/- CD25 Treg/ILC stimulation. Disclosures Levy: Heat Biologics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pelican Therapeutics: Consultancy, Research Funding.

P1.06-03 Selective Depletion of Foxp3+ Regulatory T Cells After Local Radiotherapy Induces Abscopal Effects in Murine Malignant Mesothelioma

Increasing evidence indicates that local radiotherapy (LRT) can invoke both local and systemic immune responses. Combining blockade of radiation-induced immunosuppressive responses with LRT may augment antitumor effects. Mice expressing a diphtheria toxin (DT) receptor under control of the Foxp3 locus (DEREG mice) allow conditional and efficient depletion of Foxp3+ regulatory T cell (Treg) by DT injection. This study aimed to elucidate the antitumor effect of selective depletion of Tregs combined with LRT in a murine malignant mesothelioma model. The infiltration of CD8+ T cells and Tregs was examined by flow cytometry at different time points after nonablative hypofractionated radiation (5 Gy x 3 days) in a murine mesothelioma model. AB12 murine mesothelioma cells were injected s.c. into syngeneic DEREG mice at two separate sites, defined as a “primary” site that was irradiated and a “secondary” site outside the radiation field. These mice were treated with LRT alone, DT injection alone, or a combination of LRT and DT injection. The proportion of CD8+ T cells and Tregs of total CD45+ cells in tumors were not different between radiated and untreated mice on day 2 and 5 after LRT. However, on day 7 and 12, both CD8+ T cells and Tregs in tumors significantly increased in mice treated with LRT compared with those without LRT. Selective Treg depletion by DT injection after LRT showed the best tumor growth delay in both primary and secondary tumor sites compared with LRT alone and DT injection alone. CD8+ T cells in spleen showed increased intracellular IFN-γ and Granzyme B production in mice treated with a combination of LRT and DT injection. Selective Treg depletion after LRT has the potential to evoke efficient local and systemic antitumor responses in murine mesothelioma. Our findings might have implications for future therapeutic strategies in mesothelioma patients.

Restoring the balance in autoimmunity: the promise of IL-2 mutein

Abstract The development and function of regulatory T cells (Treg) relies on interleukin-2 (IL-2), and Treg efficiently compete with other cells for IL-2 by expressing the highest levels of the IL-2 receptor alpha-chain (CD25). Accordingly, defects in Treg and the IL-2 pathway have been reported in several autoimmune diseases including type 1 diabetes (T1D). Low-dose IL-2 therapy has shown promising results in autoimmunity; however, autoreactive immune cells can also be activated by IL-2, and the pleiotropic effects of IL-2 raise concerns regarding its widespread utility. Hence, there is a need to develop novel IL-2 based therapeutics with enhanced Treg-specificity. Hypothesizing that reduced IL-2 affinity for IL-2Rβ should increase dependency on high CD25 expression and enhance Treg-selectivity, we generated and screened twenty-eight Fc-fused murine IL-2 muteins (Fc.IL-2 mutein), and identified an optimized mutein that showed reduced signaling but prolonged association with CD25. In vivo, this optimal mutein led to greater enrichment and Ki-67 induction in Treg compared to wild-type (WT) IL-2, and reduced agonism of effector cells, resulting in an improved Treg:effector ratio. In the non-obese diabetic mouse model of T1D, we observed significant protection from disease in mice treated with Fc.IL-2 mutein compared to WT IL-2. The enhanced efficacy of the mutein was most pronounced at high doses, revealing preferential Treg stimulation across a wide dose range. Thus, Fc.IL-2 mutein treatment represents a safer and more efficient alternative for the WT IL-2 therapy, allowing for selective Treg enrichment and a targeted control of the autoimmune response.

Abstract A167: Prevalence of TREG cells and effects of their inhibition on growth of oral cancer cells

Abstract Oral squamous cell carcinoma (OSCC) is one of the major cancers affecting Asian countries. The main causative factor has been tobacco habit. It has been reported that immune dysfunction in these patients is one of the major factors for tumor growth and dissemination that affects disease-free survival of the patients. We assessed the phenotypic and functional characteristics of regulatory T (Treg) CD4+CD25+FoxP3+subsets in patients with OSCC by multicoloured flow cytometry. Subsequently we investigated the effects their inhibition via TDG on growth of OSCC cell lines in vitro. An increased (p<0.05) prevalence of Treg phenotypes (CD4+CD25+, CD4+FoxP3+, CD8+FoxP3+, CD4+CD25+FoxP3+) was observed in the peripheral circulation of OSCC patients that positively correlated with clinicopathologic features. The increased frequency of CD4+CD8+CD25+FoxP3+, a unique T-cell subset, CTLA4+, GITR+, NrP1+ and granzyme B+ (GzmB) Tregs also showed a significantly higher prevalence in OSCC patients. Functionally, CD4+FoxP3+ Tregs showed skewed expression of IL2, IL10 and IL35 in patients as compared with the normal controls. Higher expression of TGFβ in tumor tissues suggests their dominant role in the up regulation of differentiation of Tregs from naive T-cells in the tumor bearing host. Further, enhanced expression of CCR5 and CCR7 on Tregs with up regulation of their ligands (CCL5, CCL19 and CCL21) in tumor cells indicates efficient recruitment and trafficking of Tregs to the tumor site. Treatment with βGBP showed growth-promoting effects on Tregs and oral cancer cells. However, the treatment with its inhibitor TDG resulted in inhibition of Treg subsets and also decreased the frequency of IL10+ and IL35+ Tregs, indicating its immunomodulatory effects. Hence, it seems reasonable to assume that modulation of functional dynamics of selective Treg subsets may be useful in enhancing antitumor immunity and developing immunotherapeutic strategies for patients with oral squamous cell carcinoma. Citation Format: Sadhna Aggarwal, Suresh C. Sharma, Satya N. Das. Prevalence of TREG cells and effects of their inhibition on growth of oral cancer cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A167.