Characterization of anti-drug antibody responses to the T-cell engaging bispecific antibody cibisatamab to understand the impact on exposure.

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.

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.