Limits...
Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms.

Pellerin A, Otero K, Czerkowicz JM, Kerns HM, Shapiro RI, Ranger AM, Otipoby KL, Taylor FR, Cameron TO, Viney JL, Rabah D - EMBO Mol Med (2015)

Bottom Line: These effects were confirmed in vivo using a single injection of 24F4A in cynomolgus monkeys. 24F4A also inhibited pDC activation by SLE-associated immune complexes (IC).In addition to the inhibitory effect of 24F4A through engagement of BDCA2, the Fc region of 24F4A was critical for potent inhibition of IC-induced IFN-I production through internalization of CD32a.This study highlights the novel therapeutic potential of an effector-competent anti-BDCA2 mAb that demonstrates a dual mechanism to dampen pDC responses for enhanced clinical efficacy in SLE.

View Article: PubMed Central - PubMed

Affiliation: Immunology Research, Biogen Idec, Cambridge, MA, USA.

Show MeSH

Related in: MedlinePlus

24F4A mediates BDCA2 internalization and type I IFN inhibition in vivoCynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A–C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).D PK/PD relationship between 24F4A serum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i–iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403047&req=5

fig03: 24F4A mediates BDCA2 internalization and type I IFN inhibition in vivoCynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A–C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).D PK/PD relationship between 24F4A serum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i–iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).

Mentions: Nine cynomolgus monkeys were divided into three groups that received a single intravenous (IV) injection of vehicle (sodium citrate buffer), 1 mg/kg 24F4A, or 10 mg/kg 24F4A. Animals were bled at various time points before and after 24F4A administration. First, we addressed whether administration of 24F4A leads to BDCA2 internalization in vivo, using flow cytometry. Because the 2D6 anti-BDCA2 clone does not cross-react with cynomolgus BDCA2 (Supplementary Table S1), a two-step approach was used to detect internalization of BDCA2 on cynomolgus monkey pDCs. Unoccupied surface BDCA2 was detected on pDCs in whole blood using fluorescently labeled 24F4A (direct method), while surface BDCA2 bound to 24F4A was detected using a fluorescently labeled anti-human IgG1 (indirect method). The lack of unoccupied BDCA2 (direct method) coupled with loss of detectable 24F4A (indirect method) indicated BDCA2 internalization. Results from a representative animal from both the vehicle-treated group and the 1 mg/kg 24F4A-treated group are shown in Fig3A and B. Prior to vehicle and 24F4A administration, the baseline surface expression of BDCA2 was assessed for each cynomolgus monkey using the direct method (Fig3A-i and B-i, dotted red line). In addition, maximal binding of BDCA2 to 24F4A was established prior to 24F4A administration by “spiking” whole blood with saturating amounts of 24F4A in vitro and measuring bound 24F4A by the indirect method (Fig3A-ii and B-ii, solid red line). Within 6 h of 24F4A administration at 1 mg/kg, BDCA2 expression on the surface of pDCs decreased to almost undetectable levels (Fig3B-iii, dotted red line) but not in the vehicle-treated group (Fig3A-iii, dotted red line). In addition, the levels of bound 24F4A (Fig3B-iv, solid black line) were indistinguishable from the vehicle-treated group (Fig3A-iv, solid black line). The lack of available BDCA2 receptor together with the lack of detectable 24F4A on the surface of pDCs indicated internalization of BDCA2. Over 95% of surface BDCA2 was internalized in all animals within 6 h of IV treatment (1 and 10 mg/kg) (Fig3C). Internalization of BDCA2 correlated with circulating levels of 24F4A, establishing a pharmacokinetic/pharmacodynamic (PK/PD) relationship in vivo. When 24F4A serum concentrations decreased to a range of 0.1–0.03 μg/ml, the level of BDCA2 recovered to > 70% of the baseline level (Fig3D-i–iii), establishing an EC50 of 0.133 μg/ml (Fig3D-iv).


Anti-BDCA2 monoclonal antibody inhibits plasmacytoid dendritic cell activation through Fc-dependent and Fc-independent mechanisms.

Pellerin A, Otero K, Czerkowicz JM, Kerns HM, Shapiro RI, Ranger AM, Otipoby KL, Taylor FR, Cameron TO, Viney JL, Rabah D - EMBO Mol Med (2015)

24F4A mediates BDCA2 internalization and type I IFN inhibition in vivoCynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A–C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).D PK/PD relationship between 24F4A serum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i–iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403047&req=5

fig03: 24F4A mediates BDCA2 internalization and type I IFN inhibition in vivoCynomolgus monkeys were administered 24F4A (10 or 1 mg/kg) or vehicle (n = 3 for each dose group) intravenously. Cynomolgus monkeys were bled at various time points, and flow cytometry was used to measure BDCA2 expression and receptor occupancy. PDCs were defined as CD20−, CD14−, CD123+, and HLADR+.A–C Prior to in vivo dosing, baseline surface levels of BDCA2 for both the vehicle (Ai) and 1 mg/kg (Bi) animals (red, dotted line) were established by staining with fluorescently labeled 24F4A (direct method). Maximal binding of 24F4A to BDCA2 was also established pre-dose in the vehicle (Aii) and 1 mg/kg (Bii) animals (red, solid line) by treating whole blood with 10 μg/ml of 24F4A at 4°C and then detecting bound 24F4A with a fluorescently labeled anti-human IgG1 (indirect method). The direct method was used to stain whole blood from both the vehicle (Aiii) and 1 mg/kg 24F4A (Biii) animals 6 h post-dose (red, dotted line). In a separate stain, the indirect method was used to detect bound 24F4A in the vehicle (Aiv) and 1 mg/kg (Biv) treated animals (black, solid line). (C) Percent BDCA2 internalization relative to pre-dose BDCA2 levels 6 h post-dose with vehicle, 10 mg/kg, or 1 mg/kg 24F4A. Graph shows mean ± standard deviation for each group (n = 3).D PK/PD relationship between 24F4A serum concentrations (red triangle, left axis) and BDCA2 expression on pDCs (black squares, right axis, normalized to pre-dose levels) from the 1 mg/kg group (i–iii). Serum 24F4A was measured by ELISA. (iv) Percent BDCA2 internalization versus serum concentration of 24F4A for all dosed cynomolgus monkeys at all time points tested.E Whole blood from vehicle- or 1 mg/kg 24F4A-treated monkeys was stimulated with CpG-A, and induction of IFN-I was measured by MxA bioassay at various time points pre- and post-treatment. Horizontal black lines represent the model-based estimates of the geometric mean of IFN-I in pre- and post-dose samples. Duplicate symbols represent independent replicates of the MxA bioassay for that time point. Statistical analysis was performed using a two-way mixed-effects analysis of variance (ANOVA).
Mentions: Nine cynomolgus monkeys were divided into three groups that received a single intravenous (IV) injection of vehicle (sodium citrate buffer), 1 mg/kg 24F4A, or 10 mg/kg 24F4A. Animals were bled at various time points before and after 24F4A administration. First, we addressed whether administration of 24F4A leads to BDCA2 internalization in vivo, using flow cytometry. Because the 2D6 anti-BDCA2 clone does not cross-react with cynomolgus BDCA2 (Supplementary Table S1), a two-step approach was used to detect internalization of BDCA2 on cynomolgus monkey pDCs. Unoccupied surface BDCA2 was detected on pDCs in whole blood using fluorescently labeled 24F4A (direct method), while surface BDCA2 bound to 24F4A was detected using a fluorescently labeled anti-human IgG1 (indirect method). The lack of unoccupied BDCA2 (direct method) coupled with loss of detectable 24F4A (indirect method) indicated BDCA2 internalization. Results from a representative animal from both the vehicle-treated group and the 1 mg/kg 24F4A-treated group are shown in Fig3A and B. Prior to vehicle and 24F4A administration, the baseline surface expression of BDCA2 was assessed for each cynomolgus monkey using the direct method (Fig3A-i and B-i, dotted red line). In addition, maximal binding of BDCA2 to 24F4A was established prior to 24F4A administration by “spiking” whole blood with saturating amounts of 24F4A in vitro and measuring bound 24F4A by the indirect method (Fig3A-ii and B-ii, solid red line). Within 6 h of 24F4A administration at 1 mg/kg, BDCA2 expression on the surface of pDCs decreased to almost undetectable levels (Fig3B-iii, dotted red line) but not in the vehicle-treated group (Fig3A-iii, dotted red line). In addition, the levels of bound 24F4A (Fig3B-iv, solid black line) were indistinguishable from the vehicle-treated group (Fig3A-iv, solid black line). The lack of available BDCA2 receptor together with the lack of detectable 24F4A on the surface of pDCs indicated internalization of BDCA2. Over 95% of surface BDCA2 was internalized in all animals within 6 h of IV treatment (1 and 10 mg/kg) (Fig3C). Internalization of BDCA2 correlated with circulating levels of 24F4A, establishing a pharmacokinetic/pharmacodynamic (PK/PD) relationship in vivo. When 24F4A serum concentrations decreased to a range of 0.1–0.03 μg/ml, the level of BDCA2 recovered to > 70% of the baseline level (Fig3D-i–iii), establishing an EC50 of 0.133 μg/ml (Fig3D-iv).

Bottom Line: These effects were confirmed in vivo using a single injection of 24F4A in cynomolgus monkeys. 24F4A also inhibited pDC activation by SLE-associated immune complexes (IC).In addition to the inhibitory effect of 24F4A through engagement of BDCA2, the Fc region of 24F4A was critical for potent inhibition of IC-induced IFN-I production through internalization of CD32a.This study highlights the novel therapeutic potential of an effector-competent anti-BDCA2 mAb that demonstrates a dual mechanism to dampen pDC responses for enhanced clinical efficacy in SLE.

View Article: PubMed Central - PubMed

Affiliation: Immunology Research, Biogen Idec, Cambridge, MA, USA.

Show MeSH
Related in: MedlinePlus