Anti-human IL20 Recombinant Antibody (2A11) (TAB-562LC) (CAT#: TAB-562LC)

Figure 1 Affinity of Patient-Derived mAbs

Binding determined by LIPS of APS1/APECED-derived mAbs and of germline counterparts to IFNa2, IFNa8, IFNa14 (19D11, 50E11, and 26B9), IL22 (35G11 and 30G1), and IL20 (20A10 and 2A11). Binding to immobilized IL17F (17E3 and 9A2) was determined by ELISA.

Meyer, S., Woodward, M., Hertel, C., Vlaicu, P., Haque, Y., Kärner, J.,... & Metsküla, K. (2016). AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies. Cell, 166(3), 582-595.

Figure 2 ELISA determination of binding of exemplary anti-IL-20 antibodies to IL-20 constructs fused either N-terminally (gl IL-20) or C-terminally (g2 IL-20) to Gaussia luciferase.

A: 2A11; B: 6E11; C: 6H2; D: 7D1; E: 20A10; F: A non-IL20 specific monoclonal antibody is used as control for non-specific binding. Antibodies 2A11, 6E11 and 20A10 bind with a high affinity to both IL-20 constructs, in contrast remaining antibodies 6H2 and 7D1 bind with a lower affinity to g2 IL-20.

Figure 3 Human-derived anti-IL-20 monoclonal antibodies neutralize recombinant IL-20-mediated STAT3 activation in HEK 293T MSR cells transiently expressing Type I and Type II IL-20 receptors.

Either cells were left untreated or stimulated with recombinant human or murine IL-20 in the absence of antibodies or in the presence of human-derived IL-20 monoclonal antibodies or a human control IgG as indicated (hulgG). Cell lysates were subjected to SDS-PAGE and pSTAT3 levels were visualized in Western blots. Total STAT3, alpha-tubulin and IL-20RB levels serve as loading control. Antibody concentration: 5 μg/ml. A: Control: rhIL-20 and rmlL-20 induce the dose-dependent phosphorylation of STAT3 in HEK 293T MSR cells transiently expressing Type I or Type II IL-20 receptors. Detection of total and phosphorylated STAT3 and IL-20RB-Myc-DDK levels. B: Stimulation with rhIL-20 (10 ng/ml) or rmIL-20 (25 ng/ml). Exemplary antibodies 2A11, 20A10 and 7D1 neutralize rhIL-20, whereas only 20A10 also potently neutralizes rmIL-20.

Figure 4 Human-derived anti-IL-20 monoclonal antibodies neutralize rhIL-20-mediated induction of KZ136-NLuc reporter constructs in HEK 293T MSR cells transiently transfected with IL-20 receptors and KZ136-NLuc.

Cells were stimulated with 120 ng/ml rhIL-20 or 200 ng/ml rmIL-20 in the presence of human-derived IL-20 monoclonal antibodies or of a control human IgG (hulgG) as indicated. rhlL-20 IC50analysis of exemplary antibodies 20A10, 7D1 and 2A11.

Figure 5 IC50 analysis of human-derived anti-IL-20 monoclonal antibodies in the chemiluminescent cellular binding assay.

HEK 293T MSR cells transiently expressing Type I or Type II IL-20 receptors were incubated with gl IL-20 supernatants in the presence of human-derived IL-20 monoclonal antibodies, A: 20A10, B: 7D1 and C: 2A11. D: IC50 values of exemplary antibodies 20A10, 7D1 and 2Al l.

Figure 6 Cross-competition

Experimental setup: 96 well microplates (Costar, USA) were coated with Rabbit-anti-GLuc specific antibody diluted 1/250 in PBS O/N at 4 °C. Plates were washed with PBS-T and blocked lh at room temperature with PBS containing 2 % BSA (Sigma, Buchs, Switzerland). 30 μL GLuc-IL-20 was added at a final concentration of 2x10⁶ LU/well and incubated for 2h at room temperature. The chimeric antibodies are premixed with the human competitor antibodies at a final concentration of 0.5 μg/ml vs. 3 μg/ml in 30 μL PBS and added to the plates. Upon incubation for 2 h at room temperature the plates were washed with PBS-T and the binding of the human-mouse chimeric antibodies was determined using a horseradish peroxidase conjugated goat anti-mouse IgG Fc-gamma specific antibody (Jackson Immuno Research, 1 :500 in 0.5 % BSA-PBS) followed by measurement of the HRP activity using a TMB substrate solution (Sigma, Buchs, Switzerland). The differential binding of exemplary human-derived anti-IL-20 monoclonal antibodies with human-mouse (hm) chimeric constructs to distinct binding sites was investigated in cross-competition experiments with 2A11 hm.

Figure 7 Cross-reactivity-Determination and comparison of binding of exemplary human-derived anti-IL-20 monoclonal antibodies 6E11, 2A11 and 20A10 to mouse and human IL-20 by GLuc-sandwich ELISA.

Figure 8 SPR analysis

Detailed analysis of the sensograms concerning the binding of human and mouse IL-20 to exemplary antibodies. A 1 : 1 binding kinetic was observed. The antigens were injected in concentrations of 1.5 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM. Calculated Affinities (KD values [M]) are indicated in the diagrams.

Figure 9 Mapping of the epitopes of the human anti-IL-20 antibodies.

Binding of antibodies to full length antigens coupled to the microarray. The Y-axis indicated the fluorescence intensity (RFU) upon detection with a Cy5-conjugated secondary antibody.