An immunodepletion procedure advances free angiopoietin-2 determination in human plasma samples during anti-cancer therapy with bispecific anti-Ang2/VEGF CrossMab.

Bispecific monoclonal IgG antibodies offer increased efficacy by antagonizing two different targets. Assessing drug mechanisms, target engagement and biomarker features, the quantification of free target levels is essential. The anti-Ang2/VEGF-CrossMab (anti-A2V) recognizes soluble vascular endothelial growth factor-A (VEGF-A) and soluble angiopoietin-2 (Ang2). However, an assay for reliable free Ang2 determination is missing. Here, we describe an immunodepletion procedure that allows for selective quantification of free Ang2 target levels by taking into advantage the bispecificity of the therapeutic antibody. The specificity for VEGF was utilized to efficiently eliminate drug-bound Ang2 from plasma samples prior to an established Ang2 measurement. The magnetic bead-based depletion procedure used an anti-idiotypic monoclonal antibody (mAb) specific for the VEGF binding site of anti-A2V (anti-Id-anti-VEGF mAb) to capture the drug along with drug-bound Ang2. High efficiencies of 99.9% were obtained for anti-A2V depletion (concentration range 300 ng/mL to 10(6)ng/mL) reflecting a 1000-fold reduction of drug-bound Ang2. A significant impact of the interaction of anti-Id-anti-VEGF mAb with anti-A2V on the Ang2 binding could be excluded. Moreover, reliable quantification of free Ang2 concentrations in plasma samples was assured by interference testing. Performing advanced free Ang2 determination including the immunodepletion step in parallel to established Ang2 measurement without immunodepletion, we compared free with total Ang2 concentrations in human plasma samples obtained from an anti-A2V Phase 1 clinical study. Samples from untreated patients displayed rather low and equal values for both free and total Ang2. In contrast, samples from drug-treated patients showed a significant reduction of free Ang2 accompanied by an accumulation in total Ang2. These results underline the value of the novel immunodepletion procedure for reliable discrimination of free vs. total target quantification with particular importance for pre-clinical and clinical development of anti-A2V. Moreover, this approach may serve as universal concept for the determination of free target levels of bispecific therapeutic antibodies.