Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

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.This work was supported by Wellcome Trust Grant 091157, JDRF International Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre, and the Medical Research Council Cusrow Wadia Fund. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (100140). U.M.N. was the recipient of a Hoffmann-La Roche postdoctoral fellowship.This is thefinal version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S089684111400146

A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.

We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation

Uptake and fate of an antibody directed against different transcytosis receptors in hCMEC/D3 monolayers.

<p><b>A–D</b> hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filters were incubated apically with 1 µg/mL of the following antibodies: the anti-human TfR antibody, 128.1 (<b>A</b>), the mouse anti–human TfR antibody, MEM-189 (<b>B</b>), an anti-human IGF-1R antibody (<b>C</b>) and the mouse anti-human insulin receptor (IR) antibody, 83–14 (<b>D</b>) for 60 min at 37°C. Luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. Cells were chased up to 5 hrs at 37°C. At the end of the chase, antibody associated with cells (▪) and media from the apical (<i>grey bars</i>) and basolateral chambers (<i>black bars</i>), were analyzed by the sensitive IgG ELISA at different time points. The total amount of IgG (▴) derived from combined value of antibody present in the lysate and in the media chambers was calculated for the entire duration of the assay. Values are means of three filters ± SEM.</p

128.1 is a high affinity antibody that competes successfully with MEM-189 for binding to TfR in ELISA and in contrast to MEM-189 shows no pH dependence.

<p><b>A</b>: Competition ELISA described in Materials and Methods showing binding of 128.1 to the extracellular subunit of TfR in the presence (▪) or absence (▴) of a pre-block by 5 µg/ml MEM-189. Similarly, binding of MEM-189 in the presence (□) or absence (▵) of 5 µg/mL 128.1. <b>B</b>, Binding ELISA described in Materials and Methods showing binding of the 128.1 anti-human transferrin receptor antibody (▪,□) and MEM-189 antibody (•,○) to the extracellular subunit of the human TfR at pH 7.4 (▪,•) or at pH 5.5 (□,○).</p

Apical to basolateral transport of ¹²⁵I-Tfn and ¹²⁵I-128.1 anti-TfR antibody in hCMEC/D3 cells.

<p><b>A</b>: Empty filters (Empty Filters) treated as described in Materials and Methods or hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filter inserts (filters with cells) (serum starved for 1 h at 37°C before assay) were incubated apically with ¹²⁵I-transferrin (Tf stock 1 µg/mL) for 60 min at 37°C. After 1 hr, radioactivity associated with media from the apical (<i>dark grey bars</i>) and basolateral chambers (<i>grey bars</i>), was analyzed in a γ counter. <b>B, C, and D</b>: hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filter inserts were serum starved for 1 h at 37°C and then incubated apically with 1 µg/mL ¹²⁵I-transferrin (in serum free medium) for 60 min at 37°C. Cells were washed to remove unbound ligand and chased up to 4 hours at 37°C (<b>B</b>) or on ice (<b>D</b>). At the end of the chase, radioactivity associated with cells (▪) and media from the apical (<i>grey bars</i>) and basolateral chambers (<i>black bars</i>), was analyzed in a gamma counter at different time points. The total amount of the radiolabelled ligand (▴) derived from combined value of radiolabel in the lysate and in the media chambers remained constant during the assay. Each data point represents values obtained from three filter membrane inserts with cells and these results are representative of three separate experiments. (<b>C</b>), denotes active protein (<i>black bars</i>), degraded protein (<i>white bars</i>) and free iodine (<i>grey bars</i>) obtained from TCA precipitation of apical or basolateral media compartments from (<b>B</b>). <b>E and F</b>: hCMEC/D3 cells grown to confluence in a contact co-culture with primary human astrocytes (<b>E</b>) or in a non-contact culture condition with the rat C6 cell line (<b>F</b>) were incubated for 1 h at 37°C in serum free media to deplete intracellular transferrin. Cultures were incubated apically with 1 µg/mL ¹²⁵I-transferrin (in serum free medium) for 60 min at 37°C. Cells were washed to remove unbound ligand and cultured at 37°C. Apical to basolateral transport of the ligand (<i>black bars</i>) or apically recycled (<i>grey bars</i>) internalised ligand was determined by analyzing the whole cell lysates (▪) and the media from the apical and basolateral chambers in a gamma counter at different time points. The total amount of the radiolabelled ligand (▴) derived from combined value of radiolabel in the lysate and in the media chambers was constant during the assay. Values are means of three filters ± SEM. <b>G and H</b>: hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filters were incubated apically with 6.5 µg/mL ¹²⁵I-128.1 (<b>G</b>) in a radiolabel assay format for 60 min at 37°C. Luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. Cells were chased up to 18 hrs at 37°C. At the end of the chase, radioactivity associated with cells (▪) and media from the apical (<i>grey bars</i>) and basolateral chambers (<i>black bars</i>), was analyzed in a gamma counter at different time points. The total amount of the antibody (▴) derived from combined value of radiolabel in the lysate and in the media chambers was calculated for the duration of the assay. Each data point represents values obtained from three filter membrane inserts with cells and these results are representative of more than three separate experiments. (<b>H</b>), denotes active protein (<i>black bars</i>), degraded protein (<i>white bars</i>) and free iodine (<i>grey bars</i>) obtained from TCA precipitation of apical or basolateral media compartments from (<b>G</b>).</p

pH dependence and uptake, fate of different antibodies directed against the human TfR in hCMEC/D3 monolayers.

<p><b>A–D</b>: Binding ELISA described in Materials and Methods showing binding of mouse anti-human transferrin receptor antibodies LT-71 (<b>A</b>), MEM-75 (<b>B</b>), M-A712 (<b>C</b>) and 13E4 (<b>D</b>) to the extracellular subunit of TfR at pH 7.4 (•) or at pH 5.5 (○). <b>E</b>–<b>I</b>: hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filters were incubated apically with 1 µg/mL of the following mouse anti-human TfR antibodies: LT-71 (<b>E</b>), MEM-75 (<b>F</b>), M-A712 (<b>G</b>), 13E4 (<b>H</b>) for 60 min at 37°C. Luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. Cells were chased up to 5 hrs at 37°C. At the end of the chase, antibody associated with cells (▪) and media from the apical (<i>white bars</i>) and basolateral chambers (<i>grey bars</i>), were analyzed by the sensitive IgG ELISA at different time points. The total amount of IgG (▴) derived from combined value of antibody present in the lysate and in the media chambers was calculated for the entire duration of the assay. Values are means of three filters ± SEM. (<b>I</b>) hCMEC/D3 cells were incubated apically with 1 µg/mL mouse anti-human TfR MEM-189 antibody as in (<b>E</b>–<b>H</b>) and cells were chased up to 5 hrs at 37°C in the presence of 50 nM Bafilomycin. At the end of the chase, antibody associated with cells (▪) and media from the apical (<i>grey bars</i>) and basolateral chambers (<i>white bars</i>), were analyzed by the sensitive IgG ELISA at different time points. The total amount of IgG (▴) derived from combined value of antibody present in the lysate and in the media chambers was calculated for the entire duration of the assay. Values are means of three filters ± SEM.</p

Description of the transcytosis assay.

<p>hCMEC/D3 cells grown to confluence on collagen and fibronectin coated membrane filter inserts (and serum starved for 1 h at 37°C before assay for experiments with ¹²⁵I-transferrin) were incubated apically with the ligand or the different antibodies for 60 min at 37°C. After 1 hr, media from the apical and basolateral chambers were collected to assess paracellular flux following which the luminal and abluminal membranes of the monolayer were washed four times with medium at RT and the washes monitored to determine efficiency of removing unbound antibody. The filters containing cells were transferred to a fresh plate containing pre-warmed medium and cells were chased up to the desired time points at 37°C or on ice and at these different time points, the ligand or antibody associated with cells or in the media from the apical and basolateral chambers was analyzed in a gamma counter or assessed by IgG ELISA.</p

Degradation of 128.1 but not MEM-189 in hCMEC/D3 cells.

<p>hCMEC/D3 endothelial cells were pulsed with 1 µg/ml 128.1 (<b>A</b>) or MEM-189 (<b>B</b>) for 10 min at 37°CThe coverslip cultures were washed and cultured at 37°C for various time periods. Cultures were fixed in 4% PFA, permeabilised and immunostained with an antibody to the late endosomal/lysosomal marker CD63 and appropriate secondary antibodies as described in Materials and Methods and examined with a laser scanning confocal microscope. Insets: secondary antibodies only.</p

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines

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 89Zr-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