Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis plays a central role in suppression of anti-tumor immunity. Blocking the axis by targeting PD-L1 with monoclonal antibodies is an effective and already clinically approved approach to treat cancer patients. Glyco-engineering technology can be used to optimize different properties of monoclonal antibodies, for example, binding to FcγRs. We generated two glycosylation variants of the same anti-PD-L1 antibody: one bearing core fucosylated N-glycans in its Fc part (92%) and its de-fucosylated counterpart (4%). The two glycosylation variants were compared to a non-glycosylated commercially available anti-PD-L1 antibody in various assays. No differences were observed regarding binding to PD-L1 and blocking of this interaction with its counter receptors PD-1 or CD80. The de-fucosylated anti-PD-L1 antibody showed increased FcγRIIIa binding resulting in enhanced antibody dependent cellular cytotoxicity (ADCC) activity against PD-L1+ cancer cells compared to the “normal”-glycosylated variant. Both glycosylation variants showed no antibody-mediated lysis of B cells and monocytes. The non-glycosylated reference antibody showed no FcγRIIIa engagement and no ADCC activity. Using mixed leukocyte reaction it was observed that the de-fucosylated anti-PD-L1 antibody induced the strongest CD8 T cell activation determined by expression of activation markers, proliferation, and cytotoxicity against cancer cells. The systematic comparison of anti-PD-L1 antibody glycosylation variants with different Fc-mediated potencies demonstrates that our glyco-optimization approach has the potential to enhance CD8 T cell-mediated anti-tumor activity which may improve the therapeutic benefit of anti-PD-L1 antibodies

Dataset on the activation of Muller cells through macrophages upon hypoxia in the retina

The dataset presented in this article complements the article entitled “Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells” (C. Nürnberg, N. Kociok, C. Brockmann, T. Lischke, S. Crespo-Garcia, N. Reichhart, S. Wolf, R. Baumgrass, S.A. Eming, S. Beer- Hammer, and A.M. Joussen). This complementary dataset provides further insight into the experimental validation of the VEGFfl/fl LysMCre (here named VEGFmcko) knockout model used in the main article through genomic and quantitative Real-Time PCR in various murine tissues as well as additional flow cytometry data and immunohistochemical stainings. By providing these data, we aim to enable researcher to reproduce and critically analyze our data

data_sheet_1_Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts.docx

<p>The programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis plays a central role in suppression of anti-tumor immunity. Blocking the axis by targeting PD-L1 with monoclonal antibodies is an effective and already clinically approved approach to treat cancer patients. Glyco-engineering technology can be used to optimize different properties of monoclonal antibodies, for example, binding to FcγRs. We generated two glycosylation variants of the same anti-PD-L1 antibody: one bearing core fucosylated N-glycans in its Fc part (92%) and its de-fucosylated counterpart (4%). The two glycosylation variants were compared to a non-glycosylated commercially available anti-PD-L1 antibody in various assays. No differences were observed regarding binding to PD-L1 and blocking of this interaction with its counter receptors PD-1 or CD80. The de-fucosylated anti-PD-L1 antibody showed increased FcγRIIIa binding resulting in enhanced antibody dependent cellular cytotoxicity (ADCC) activity against PD-L1⁺ cancer cells compared to the “normal”-glycosylated variant. Both glycosylation variants showed no antibody-mediated lysis of B cells and monocytes. The non-glycosylated reference antibody showed no FcγRIIIa engagement and no ADCC activity. Using mixed leukocyte reaction it was observed that the de-fucosylated anti-PD-L1 antibody induced the strongest CD8 T cell activation determined by expression of activation markers, proliferation, and cytotoxicity against cancer cells. The systematic comparison of anti-PD-L1 antibody glycosylation variants with different Fc-mediated potencies demonstrates that our glyco-optimization approach has the potential to enhance CD8 T cell-mediated anti-tumor activity which may improve the therapeutic benefit of anti-PD-L1 antibodies.</p

Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Miller cells

Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Muller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Muller cells

CD38 Is Expressed on Inflammatory Cells of the Intestine and Promotes Intestinal Inflammation

<div><p>The enzyme CD38 is expressed on a variety of hematopoietic and non-hematopoietic cells and is involved in diverse processes such as generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Here, we show that under homeostatic conditions CD38 is highly expressed on immune cells of the colon mucosa of C57BL/6 mice. Myeloid cells recruited to this tissue upon inflammation also express enhanced levels of CD38. To determine the role of CD38 in intestinal inflammation, we applied the dextran sulfate sodium (DSS) colitis model. Whereas wild-type mice developed severe colitis, CD38^-/- mice had only mild disease following DSS-treatment. Histologic examination of the colon mucosa revealed pronounced inflammatory damage with dense infiltrates containing numerous granulocytes and macrophages in wild-type animals, while these findings were significantly attenuated in CD38^-/- mice. Despite attenuated histological findings, the mRNA expression of inflammatory cytokines and chemokines was only marginally lower in the colons of CD38^-/- mice as compared to wild-type mice. In conclusion, our results identify a function for CD38 in the control of inflammatory processes in the colon.</p></div

Expression of CD38 on T cells.

<p>Wild-type and CD38^-/- mice received 3% DSS in the drinking water or were left untreated (wo). After 5 days, DSS water was replaced by normal tap water. On day 7, cells were isolated from spleen as well as large intestine epithelium and lamina propria (LP) and analyzed by flow cytometry. Blots show CD38 expression on viable CD45⁺ CD4⁺ and CD8α⁺ T cells. For the spleen, only CD8αβ⁺ T cells are shown. Viable CD45⁺CD19⁺ B cells are only presented for the spleen. For the large intestine, CD8α⁺ T cells are further separated into conventional CD8αβ⁺ and unconventional CD8αα⁺ (CD8α⁺β^-) T cells. Dot blots give representative results for cells pooled from 5 mice per group.</p

CD38 Is Expressed on Inflammatory Cells of the Intestine and Promotes Intestinal Inflammation - Table 1

<p>CD38 Is Expressed on Inflammatory Cells of the Intestine and Promotes Intestinal Inflammation - Table 1 </p

Morphological changes in the colons of wild-type and CD38^-/- mice after DSS treatment.

<p>Wild-type and CD38^-/- mice were treated with DSS as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126007#pone.0126007.g001" target="_blank">Fig 1</a>. On day 7, mice were killed and colon sections were H&E stained. (A) Representative H&E stained sections of colons from DSS-treated wild-type (a, a’) and CD38^-/- mice (b, b’). Original magnification: 200×. (B). Colon sections were evaluated for tissue damage (score 0–4) and for cellular infiltration (score 0–4), and both scores were added up to a histological score. Parameters of scoring are given in the method section. The figure gives results for individual mice and the median of 6 mice per group. * p<0.05.</p

CD38^-/- mice develop only mild colitis in response to DSS-treatment.

<p>Wild-type and CD38^-/- mice were treated with DSS as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126007#pone.0126007.g001" target="_blank">Fig 1</a>. On day 7, mice were killed and colons were analyzed. (A) Weight changes of wild-type and CD38^-/- mice after DSS treatment. Weight was determined daily and is given as % of weight one day before the beginning of DSS treatment. Results represent the mean ± SEM of 6 individually analyzed mice per group. (B) Relative colon length of untreated and DSS-treated wild-type and CD38^-/- mice. Colon length was normalized to the whole-animal body weight, which was determined before DSS treatment. Bars give the mean ± SEM of 5 or 6 individually analyzed mice per group. (C) Clinical score of DSS-treated wild-type and CD38^-/- mice at day 7. Parameters of scoring are given in the method section. Values for individually analyzed mice and the median are shown. * p<0.05; ns not significant (p>0.05)</p