A recombinant heavy chain antibody approach blocks ART2 mediated deletion of an iNKT cell population that upon activation inhibits autoimmune diabetes

Abstract

The ectoenzyme ADP-ribosyltransferase 2.2 (ART2.2) can apoptotically delete various T-cell subsets. Depending on the involved apoptotic T-cell subset, enhanced ART2.2 activity could result in immunosuppression or autoimmunity. Diminished activity of the CD38 ectoenzyme that normally represents a counter-regulatory competitor for the NAD substrate represents one mechanism enhancing ART2.2 activity. Hence, it would be desirable to develop an agent that efficiently blocks ART2.2 activity in vivo. While the llama derived recombinant s+16 single domain antibody overcame the difficulty of specifically targeting the ART2.2 catalytic site potential therapeutic use of this reagent is limited due to short in vivo persistence. Thus, we tested if a modified version of s+16 incorporating the murine IgG1 Fc tail (s+16Fc) mediated long-term efficient in vivo suppression of ART2.2. We reasoned an ideal model to test the s+16Fc reagent were NOD mice in which genetic ablation of CD38 results in an ART2.2 mediated reduction in already sub-normal numbers of immunoregulatory natural killer T-(NKT) cells to a level that no longer allows them when activated by the super-agonist alpha-galactosylceramide (alpha-GalCer) to elicit effects inhibiting autoimmune type 1 diabetes (T1D) development. Treatment with s+16Fc efficiently mediated long-term in vivo inhibition of ART2.2 activity in NOD.CD38(null) mice, restoring their iNKT cell numbers to levels that upon alpha-GalCer activation were capable of inhibiting T1D development.

Fig. 1. ART2 and P2X7 levels on iNKT cells at various developmental stages and comparison of iNKT cell numbers and ART2 activity in NOD and NOD.CD38^null mice

a) Total thymocytes and splenocytes from NOD mice were stained with antibodies specific for either ART2, P2X7, or CD3 and an iNKT TCR binding CD1d-tetramer and analyzed by flow cytometry. Gating was on conventional T-cells (tetramer negative) or iNKT cells. For P2X7 analyses staining with pre-immune serum and secondary antibodies is also shown for CD3+ cells. Data are representative profiles of multiple independent observations. b) Proportions and ART2 expression levels of splenic iNKT cells in the indicated strains. Data are representative profiles of multiple independent observations. c) ART2 enzymatic activity of splenic iNKT cells from the indicated strains. Splenocytes were incubated with or without 1μg S+16 for 15 minutes followed by treatment with the etheno-NAD analog for 10 minutes at 4°C. Cells were washed and stained with a specific fluorochrome conjugated mAb (1G4) that detects eADP-ribosylated cell surface proteins and an iNKT TCR binding CD1d-tetramer and analyzed by flow cytometry. Data are representative profiles of multiple independent observations.

Fig. 2. Titration and serum persistence of the S+16Fc heavy chain ART2 blocking antibody

a) NOD.CD38^null mice were injected i.p with indicated amounts of S+16Fc. After 24 h dispersed lymph node cells were incubated with or without etheno-NAD for 10 minutes at 4°C, washed, stained with 1G4-mAb that detects eADP-ribosylated cell surface proteins and analyzed by flow cytometry. White and gray histograms respectively indicate incubation with or without etheno-NAD. Data are representative of 3 independent experiments. b) NOD.CD38^null mice were injected i.p. with 5μg S+16Fc and sacrificed at the indicated timepoints. Spontaneous NICD was assessed by Annexin V staining after a 30 min incubation at 4°C (panel 2) or 37°C (panels 1,3-5). Data are representative of 3 independent experiments.

Fig. 3. Enhanced survival of peripheral iNKT cells in NOD.CD38^null mice after long term treatment with s+16Fc

NOD.CD38^null mice were injected i.p. once weekly with 5μg of S+16mFc-LSF. At the indicated time points PLN cells and splenocytes were assessed by flow cytometry for proportions (a) and numbers (b) of iNKT cells by tetramer analyses (n=4-6 per time point). Differences in proportions and numbers were compared by One-Way Analysis of Variance. c) Representative flow cytometric profiles of proportions of PLN origin iNKT cells expressing CD4 in NOD controls and NOD.CD38^null mice that did or did not receive four once weekly s+16Fc injections. Gating is on iNKT cells identified by positive tetramer staining. d) Mean ratio over time of CD4+/DN iNKT-cells in NOD.CD38^null mice receiving long term s+16Fc treatment (n=3 to 6 per group). Differences in ratios were compared by One-Way Analysis of Variance. Errorbars represent mean + SEM.

Fig. 4. Long term blocking of ART2 enzymatic activity restores iNKT — DC signaling axis in NOD.CD38^null mice

NOD.CD38^null mice were injected once weekly for 4 weeks with 5μg s+16Fc. Untreated standard NOD and NOD.CD38^null mice served as controls. Subsequently, mice in each group were then injected i.p. with 2 μg a-GalCer or vehicle (DMSO). Numbers of dendritic cells (a) and iNKT cells (b) within PLNs of mice in each treatment group were assessed 4 days later by flow cytometry. In panel b, n=4 per group. Differences in cell numbers were compared by One-Way Analysis of Variance. Errorbars represent mean + SEM.

Fig. 5. Long term blocking of ART2 enzymatic activity restores ability of alpha-GalCer to protect CD38 deficient NOD from adoptively transferred T1D

NOD.CD38^null mice were injected i.p. once weekly for 7 weeks with 5 μg s+16Fc or control antibody (l−15Fc). For the last 4 weeks mice were additionally also injected i.p. with 2 μg a-Galcer. One group of mice received the a-Galcer injections only and then a single shot of s+16Fc just before irradiation. Mice were then sublethally irradiated (600R) and injected i.v. with 5×10⁶ NOD.Rag1^null.AI4 splenocytes. (a) Urinary glucose was monitored daily to determine T1D onset. Incidence curves were compared according to the Logrank Test. (b) At time of T1D onset or day 14 after transfer mice were sacrificed and numbers of AI4 CD8 T-cells and iNKT cells in PLNs were determined by flow cytometry (n=5 per group). Differences in numbers were compared using Wilcoxon rank sum test. Errorbars represent mean + SEM.

Fig. 6. Combination s+16Fc and α-GalCer treatment allows Treg expansion in the PLNs of NOD.CD38^null mice after AI4 T cell transfer

(a) NOD.CD38^null mice (n=5 per group) were treated once weekly for 7 weeks with 5 μg s+16Fc. For the last 4 weeks mice were additionally injected with 2 μg α-GalCer. Other groups were treated for 4 weeks with α-GalCer or DMSO only. Mice were then sublethally irradiated (600 R) and infused with 5×10⁶ NOD.Rag1^null.AI4 splenocytes. At day 4 post-AI4 T cell transfer proportions and numbers of phenotypic CD4+CD25+FoxP3+ Tregs in PLNs and spleen were assessed by flow cytometry. Differences in percentage and numbers were analyzed using Wilcoxon rank sum test. Errorbars represent mean + SEM. (b, c) CD4+CD25− T-cells from NOD spleens were labeled with CFSE and incubated for 3 days with 5μg/ml anti-CD3 in the presence or absence of different ratios of putative CD4+CD25+ Tregs purified from spleens of NOD or NOD.CD38^null mice. One group of NOD.CD38^null mice were injected with 5μg s+16Fc 24 hours prior to purification of Tregs. CFSE dilution was measured by flow cytometry to assess proliferation of responder cells. Suppression assays were done in technical triplicates. (b) Treg activity shown as percent suppression of responder cell proliferation in the absence of Tregs. Responder cell to Treg ratios are indicated. Results are representative of 3 independent experiments. (c) Representative flow cytometric profiles of responder cell CSFE dilution in the presence of the indicated sources of Tregs at a 1:1 ratio.

Fig. 7. Long-term treatment with s+16Fc alone does not inhibit spontaneous T1D development in NOD.CD38^null mice, but restores their ability to respond to α-GalCer in a disease protective manner

(a) NOD.CD38^null males and standard NOD females were treated with weekly i.p. injections starting after weaning with 5μg of the s+16Fc ART2.2 blocking or the control antibody (l−15Fc). Urinary glucose levels were monitored weekly to determine spontaneous T1D onset. (b) Mice were sacrificed at T1D onset and numbers of viable iNKT cells in their PLNs were determined. (c) NOD.CD38^null females were injected with vehicle control or 2μg of a-GalCer once weekly from 6 weeks of age. A group of NOD.CD38^null mice were pretreated once weekly for 3 weeks with 5μg of the ART2.2 blocking s+16Fc with the treatment then continuing in combination with the a-GalCer injections. Urinary glucose levels were monitored weekly to determine spontaneous T1D onset. Incidence curves were compared according to the Logrank Test.