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Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy., Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352)., Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro ., Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed., Competing Interests: Authors AI, JB, CK, PU, NJ, FH, LF, EO-H, IW, CB, RFS, VK and SF are or were employees of F. Hoffmann-La Roche Ltd. Author AS discloses employment Princess Margaret Cancer Centre, University Health Network, research funding Alkermes, ALX Oncology, Amgen, Array Biopharma/Pfizer, AstraZeneca/Medimmune, Bayer, BMS, Genentech, GSK, Janssen Oncology/Johnson & Johnson, Merck, Northern Biologics, Novartis, Nubiyota, Oncorus, Roche, Regeneron, Seagen, Servier, Surface Oncology, Symphogen, Treadwell, and advisory committees BMS, Merck. Author EM discloses honoraria BMS, MSD, Novartis, Pierre Fabre, Sanofi and advisory committees and speaker’s bureau BMS, MSD, Novartis, Pierre Fabre, Sanofi. Author GA-Y discloses research funding AstraZeneca, Roche/Genentech. Author OB discloses advisory committees BMS, MSD, Pierre Fabre, Sanofi. Author IS discloses consultancy IO Biotech, MSD, Novartis, Pierre Fabre, TILT Biotherapeutics, research funding Adaptimmune, Asgard Biotech, Enara Bio, Evaxion Biotech, IO Biotech, Lytix Biopharma, TILT Biotherapeutics, honoraria BMS, MSD, Novartis, Novo Nordisk, Pierre Fabre, Sanofi Aventis, Takeda, patents and royalties IO Biotech, and investigator for initiated clinical trial BMS. Author MS discloses consultancy BMS, Numab, research funding Roche, advisory committees Numab, and speaker’s bureau BMS, MSD. Author VG discloses research funding Bayer, Boehringer, Roche and institutional funding Amcure, Astelas, Astra Zeneca, Bayer, BeiGene, BMS, FibroGen, Genentech, Lilly, Medimmune, Merck Serono, MSD, Natera, Novartis, Roche, Servier, Sierra Oncology, Takeda. Author MM discloses consultancy Castle Biosciences, Eisai, IQVIA, Merck, Moderna, Pfizer and research funding Aadi Biosciences, Alpine Immune Sciences, Arcus Biosciences, Arvinas, Ascentage Pharma Group, ASCO, Astellas, Aulos Bioscience, Bayer, Bicycle Therapeutics, Biomed Valley Discoveries, BioNTech, BMS, C4 Therapeutics, Dragonfly Therapeutics, EMD Serono, Epizyme, Erasca, Exelixis, Foghorn Therapeutics, G1 Therapeutics, Genentech/Roche, Gilead Sciences, GSK, IDEAYA Biosciences, Ikena Oncology, ImmVira Pharma, Infinity Pharmaceuticals, Jacobio Pharmaceuticals, Kechow Pharma, Kezar Life Sciences, Kinnate BioPharma, MedImmune, Mereo BioPharma, Metabomed, Moderna, NBE Therapeutics, Nektar, Novartis, NucMito Pharmaceuticals, OncoC4, Oncorus, OnKure, PACT Pharma, Pfizer, Plexxikon, Poseida, Prelude Therapeutics, Pyramid Biosciences, Regeneron, Sapience Therapeutics, Scholar Rock, Seattle Genetics, Synthrox, Tenebio, Tempest Therapeutics, Tizona Therapeutics, TMUNITY Therapeutics, TopAlliance Biosciences, Xilio. Author MC discloses consulting and advisory boards BMS, AstraZeneca, Immunocore, Regeneron, Merck. Author RD discloses consulting and/or advisory relationships Alligator, Amgen, Axivox, BMS, Catalym, MSD, Novartis, Pfizer, Pierre Fabre, Regeneron, Roche, Simcere, Sanofi, Second Genome, Sun Pharma, Takeda, TouchIME, T3 Pharma. Authors CK, LF, IW, and VK disclose employment, equity, and patents and royalties Roche. Authors NJ and EO-H disclose employment Roche. Authors JB, FH, CB, RS, PU, AI and SF discloses employment and equity Roche. The Reviewer FD declared a past co-authorship with the author RD to the handling editor. The authors declare that this study received funding from F. Hoffman-La Roche Ltd. F. Hoffmann-La Roche Ltd was involved in the study design, collection, analysis, interpretation of data, the writing of this article, and the decision to submit it for publication., (Copyright © 2024 Spreafico, Couselo, Irmisch, Bessa, Au-Yeung, Bechter, Svane, Sanmamed, Gambardella, McKean, Callahan, Dummer, Klein, Umaña, Justies, Heil, Fahrni, Opolka-Hoffmann, Waldhauer, Bleul, Staack, Karanikas and Fowler.)

TYRP1-TCB is a CD3 T-cell bispecific (CD3-TCB) antibody for the treatment of advanced melanoma. A tumor growth inhibition (TGI) model was developed using mouse xenograft data with TYRP1-TCB monotherapy or TYRP1-TCB plus anti-PD-L1 combination. The model was translated to humans to inform a refined clinical strategy. From xenograft mouse data, we estimated an EC 50 of 0.345 mg/L for TYRP1-TCB, close to what was observed in vitro using the same tumor cell line. The model showed that, though increasing the dose of TYRP1-TCB in monotherapy delays the time to tumor regrowth and promotes higher tumor cell killing, it also induces a faster rate of tumor regrowth. Combination with anti-PD-L1 extended the time to tumor regrowth by 25% while also decreasing the tumor regrowth rate by 69% compared to the same dose of TYRP1-TCB alone. The model translation to humans predicts that if patients' tumors were scanned every 6 weeks, only 46% of the monotherapy responders would be detected even at a TYRP1-TCB dose resulting in exposures above the EC 90 . However, combination of TYRP1-TCB and anti-PD-L1 in the clinic is predicted to more than double the overall response rate (ORR), duration of response (DoR) and progression-free survival (PFS) compared to TYRP1-TCB monotherapy. As a result, it is highly recommended to consider development of CD3-TCBs as part of a combination therapy from the outset, without the need to escalate the CD3-TCB up to the Maximum Tolerated Dose (MTD) in monotherapy and without gating the combination only on RECIST-derived efficacy metrics., (© 2022. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs., (©2022 American Association for Cancer Research.)

Expansion and differentiation of antigen-experienced PD-1 + TCF-1 + stem-like CD8 + T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade 1-4 . Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8 + T cells similar to those generated in an acute infection 5 . IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor β- and γ-chain (IL-2Rβγ)-biased agonists are currently being developed 6-10 . Here we show that IL-2Rβγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell recovers the ability to differentiate stem-like CD8 + T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections., (© 2022. The Author(s).)

Monoclonal antibodies play an important role in the treatment of various diseases. However, the development of these drugs against neurological disorders where the drug target is located in the brain is challenging and requires a good understanding of the local drug concentration in the brain. In this original research, we investigated the systemic and local pharmacokinetics in the brain of healthy rats after either intravenous (IV) or intracerebroventricular (ICV) administration of EGFRvIII-T-Cell bispecific (TCB), a bispecific monoclonal antibody. We established an experimental protocol that allows serial sampling in serum, cerebrospinal fluid (CSF) and interstitial fluid (ISF) of the prefrontal cortex in freely moving rats. For detection of drug concentration in ISF, a push-pull microdialysis technique with large pore membranes was applied. Brain uptake into CSF and ISF was characterized and quantified with a reduced brain physiologically-based pharmacokinetic model. The model allowed us to interpret the pharmacokinetic processes of brain uptake after different routes of administration. The proposed model capturing the pharmacokinetics in serum, CSF and ISF of the prefrontal cortex suggests a barrier function between the CSF and ISF that impedes free antibody transfer. This finding suggests that ICV administration may not be better suited to reach higher local drug exposure as compared to IV administration. The model enabled us to quantify the relative contribution of the blood-brain barrier (BBB) and Blood-CSF-Barrier to the uptake into the interstitial fluid of the brain. In addition, we compared the brain uptake of three monoclonal antibodies after IV dosing. In summary, the presented approach can be applied to profile compounds based on their relative uptake in the brain and provides quantitative insights into which pathways are contributing to the net exposure in the brain., Competing Interests: AV, IW, MO, and A-CW were employed by F. Hoffmann-La Roche Ltd. TK and AA-B were employed by Roche Diagnostics GmbH at the time of submission of this manuscript. A-CW, IW, MO, TK, and AA-B are stockholders of F. Hoffmann-La Roche Ltd. MH was employed by and is stockholder of Charles River Laboratories at the time of submission of this manuscript., (Copyright © 2022 Van De Vyver, Walz, Heins, Abdolzade-Bavil, Kraft, Waldhauer and Otteneder.)

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rβγ > IL-2 Rβγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rβγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89 Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo , CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8 + T cells, skewing the CD8 + :CD4 + ratio toward CD8 + T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting., Competing Interests: All authors except for Ramona Schlenker were salaried employees of Roche Glycart AG or InSphero AG when the work was performed. No other potential conflict of interest was reported. Ramona Schlenker has no conflict of interest to declare.

Purpose: Imgatuzumab (GA201) is a novel anti-EGFR mAb that is glycoengineered for enhanced antibody-dependent cellular cytotoxicity (ADCC). Future treatment schedules for imgatuzumab will likely involve the use of potentially immunosuppressive drugs, such as premedication therapies, to mitigate infusion reactions characteristic of mAb therapy and chemotherapy combination partners. Because of the strong immunologic component of mode of action of imgatuzumab, it is important to understand whether these drugs influence imgatuzumab-mediated ADCC and impact efficacy., Experimental Design: We performed a series of ADCC assays using human peripheral blood mononuclear cells that were first preincubated in physiologically relevant concentrations of commonly used premedication drugs and cancer chemotherapies. The ability of common chemotherapy agents to enhance the efficacy of imgatuzumab in vivo was then examined using orthotopic xenograft models of human cancer., Results: A majority of premedication and chemotherapy drugs investigated had no significant effect on the ADCC activity of imgatuzumab in vitro Furthermore, enhanced in vivo efficacy was seen with imgatuzumab combination regimens compared with single-agent imgatuzumab, single-agent chemotherapy, or cetuximab combinations., Conclusions: These data indicate that medications currently coadministered with anti-EGFR therapies are unlikely to diminish the ADCC capabilities of imgatuzumab. Further studies using syngeneic models with functional adaptive T-cell responses are now required to fully understand how chemotherapy agents will influence a long-term response to imgatuzumab therapy. Thus, this study and future ones can provide a framework for designing imgatuzumab combination regimens with enhanced efficacy for investigation in phase II trials. Clin Cancer Res; 22(10); 2453-61. ©2015 AACR., (©2015 American Association for Cancer Research.)

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR., (©2016 American Association for Cancer Research.)

Unlabelled: One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA., Methods: SPECT and PET with (111)In- and (89)Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score., Results: Both (111)In-28H1 and (89)Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of (111)In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis., Conclusion: SPECT/CT imaging with the anti-FAP antibody (111)In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. (89)Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of (89)Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)